Polypeptide

ABSTRACT

Canine and feline 5T4 polypeptide sequences and nucleotide sequences encoding them are provided. A vector system comprising a nucleic acid encoding 5T4 and a 5T4-specific agent are also provided.

FIELD OF THE INVENTION

[0001] The present invention relates to 5T4 antigens, 5T4-specificagents and their use in immunotherapy.

BACKGROUND TO THE INVENTION

[0002] Tumours are relatively common in companion animals (see Mailtoand Lagadic (1990) Recueil de Medacine. Veterinaire Special Cancerologiep937-947 for a descriptive epidemiology of canine and feline tumours).

[0003] Current methods for treating tumours in cats and dogs includesurgery, chemotherapy and radiotherapy. These methods are associatedwith a number of disadvantages. For example, they all involve a highlevel of trauma for the patient and they are not always effective ineliminating all the cancerous cells.

[0004] An alternative therapeutic approach is immunotherapy whichinvolves the specific or non-specific stimulation of immune reactions ofthe patient in order to promote the immunological rejection of cancerouscells. There are several methods of immunotherapy:

[0005] Non-specific immunotherapy—non-specific stimulation of the immunesystem (e.g. by using an agent which acts like an adjuvant).

[0006] Specific Passive immunotherapy (serotherapy)—tranfserringanti-tumour antibodies to the patient.

[0007] Adoptive immunotherapy—transferring immunocompetent allogeniccells from a healthy individual (eg bone marrow cells)

[0008] Specific active immunotherapy—stimulating the immune defences ofthe cancer patient by providing the antigens associated with the tumour(e.g. using irradiated cancer cells)

[0009] Immunotherapy may be a method of complementary treatment used incombination with surgery, chemotherapy and/or radiotherapy. A study ofimmunotherapeutic chemical trials in cats and dogs is given in Hayes(1990) Recueil de Medicine Veterinaire 16(11).

[0010] An immunotherapeutic approach may be directed against an antigenwhich is peculiar to the tumour. One strategy for canine and felinecancer immunotherapy would be to identify a tumour-associated antigens(TAAs), expressed on cat or dog tumours, useful for eliciting ananti-tumour immunotherapeutic response.

SUMMARY OF THE INVENTION

[0011] The present inventors have shown that a significant proportion ofcanine and feline tumours express an oncofetal leucine-richglycoprotein, known as “5T4”. There is also presented for the first timethe full protein and nucleotide sequences for canine 5T4 and feline 5T4.

[0012] The first aspect of the invention relates to canine and feline5T4 sequences.

[0013] In this aspect, the present invention provides

[0014] i) a canine 5T4 polypeptide having the amino acid sequence shownin SEQ ID No 1 or a variant, homologue, fragment or derivative thereof;and

[0015] ii) a nucleotide sequence capable of encoding such a canine 5T4polypeptide. Preferably the nucleotide sequence has the sequence shownas SEQ ID NO 2 or a variant, homologue, fragment or derivative thereof.

[0016] The present invention also provides

[0017] i) a feline 5T4 polypeptide having the amino acid sequence shownin SEQ ID No 3 or a variant, homologue, fragment or derivative thereof;and

[0018] ii) a nucleotide sequence capable of encoding such a feline 5T4polypeptide. Preferably the nucleotide sequence has the sequence shownas SEQ ID NO 4 or a variant, homologue, fragment or derivative thereof.

[0019] The present invention also provides an agent specific to a canineor feline 0.5T4 nucleotide sequence. For example, the agent may comprisean antisense sequence capable of binding specifically to the 5T4sequence.

[0020] In a second aspect, the present invention provides a vectorsystem expressing a polynucleotide encoding a canine or feline 5T4antigen.

[0021] Expression of 5T4 antigen in a subject elicits animmunotherapeutic anti-tumour response. Preferably, the viral vectorfavours CTL responses to expressed antigens, and is advantageously apoxvirus vector, such as a vaccinia virus vector. Further vectors, bothviral and non-viral, which are suitable for delivering 5T4 antigen aredescribed below.

[0022] In a third aspect, the invention provides an agent capable ofbinding specifically to canine or feline 5T4 protein. The agent may bean antibody. For example, the agent may be an antibody raised againstthe canine or feline 5T4 protein (or fragment thereof) of the firstaspect of the invention.

[0023] The present invention also provides to the sequential use of avector encoding a 5T4 antigen and such an anti-5T4 agent. Where theanti-5T4 agent is a protein (such as an antibody or derivative thereof)it may be administered as naked DNA (for example, in a plasmid), or inan expression vector (which may be viral or non-viral) or directly in aprotein form. The agent may be fused with an immunostimulatory molecule.

[0024] Thus, the invention provides a kit comprising a vector accordingto the second aspect of the invention and an agent according to thethird aspect of the invention, for simultaneous, separate, or sequentialuse, preferably for use in the treatment of tumours.

[0025] In a fourth aspect the present invention provides a vaccine,priming composition or boosting composition comprising such apolypeptide, polynucleotide, vector system or agent. The vaccine,priming or boosting composition may comprise one or more adjuvants.

[0026] It has been found that multiple-dose procedures are often moreeffective at generating an immune response that a single administrationof vaccine. Prime-boost regimes may be homologous (where the samecomposition is administered two or more times) or heterologous.

[0027] An example of a heterologous prime-boost regime would be theadministration of at least one dose of a DNA vaccine, followed by atleast one dose of a viral vaccine. An example of a homologousprime-boost regime is repeated doses of a viral vector system.

[0028] In this respect, the present invention also comprises a kit whichcomprises:

[0029] a first composition comprising a polynucleotide encoding a 5T4antigen, and

[0030] a second composition comprising a vector system according to thesecond aspect of the invention

[0031] for simultaneous, separate or sequential administration to asubject.

[0032] The first composition may, for example, be a naked DNA vector.

[0033] In a fifth aspect, the present invention provides the use of sucha 5T4 antigen, polypeptide, polynucleotide, vaccine, primingcomposition, boosting composition or kit in the manufacture of amedicament for the prevention and/or treatment of a disease in asubject.

[0034] The present invention also provides a method for the treatmentand/or prevention of a disease in a subject which comprises the step ofadministration of such a 5T4 antigen, protein, polynucleotide, vaccine,priming composition, boosting composition to the subject.

[0035] Preferably the method of the fifth aspect of the invention is amethod for the immunotherapy of a tumour in a subject.

DETAILED DESCRIPTION OF THE INVENTION

[0036] Other aspects of the present invention are presented in theaccompanying claims and in the following description and drawings. Theseaspects are presented under separate section headings. However, it is tobe understood that the teachings under each section are not necessarilylimited to that particular section heading.

[0037] 5T4 Proteins

[0038] The first aspect of the invention relates to canine and feline5T4 proteins and their associated polynucleotides.

[0039] In humans, the oncofetal leucine-rich glycoprotein, 5T4, isexpressed by a wide variety of carcinomas, but on normal adult tissuesexpression is restricted to the placenta with low levels also beingfound on a few specialised epithelia. Presence of the antigen on cancercells is associated with metastasis and has been shown to be anindependent indicator of poor prognosis in a number of differentcancers.

[0040] The human tumour-associated antigen 5T4 is a 72 kDa glycoproteinand has been characterised (for example, in WO89/07947). The fullnucleic acid sequence of human 5T4 is known (iGenBank accession no.Z29083; Myers et al., 1994 J Biol Chem 169: 9319-24).

[0041] WO 00/29428 describes the partial sequence of canine 5T4.

[0042] The present invention provides, for the first time, the fullamino acid and nucleic acid sequences for canine and human 5T4. Despitethe fact the human and partial canine sequences were available,isolation and cloning of the full canine and feline sequences was notstraightforward. In this respect a number of attempts were made toisolate the canine gene by PCR using primers based on the human sequenceimmediately outside the coding region. Such attempts failed due tounexpected differences between the human and canine sequencesimmediately upstream of the start codon.CCCAGCTCCGGGGAGCGCCGCGCCGCGCCGCGATG Canine    AGCTCCGGGGAAACGCGAGCCfailed PRIMER CCCAGCTCCGGGGAAACGCGAGCCGCGATG Human

[0043] Amino Acid Sequences

[0044] As used herein, the term “amino acid sequence” refers to peptide,polypeptide sequences, protein sequences or portions thereof.

[0045] The present invention covers variants, homologues or derivativesof the amino acid sequences presented herein, as well as variants,homologues or derivatives of the nucleotide sequence coding for thoseamino acid sequences.

[0046] In the context of the present invention, a homologous sequence istaken to include an amino acid sequence which is at least 75, 85 or 90%identical, preferably at least 95 or 98% identical at the amino acidlevel over at least, for example, the amino acid sequence as set out inSEQ ID No 1 or SEQ ID No 3 of the sequence listing herein. Inparticular, homology should typically be considered with respect tothose regions of the sequence known to be essential for bindingspecificity (such as amino acids at positions) rather than non-essentialneighbouring sequences. Although homology can also be considered interms of similarity (i.e. amino acid residues having similar chemicalproperties/functions), in the context of the present invention it ispreferred to express homology in terms of sequence identity.

[0047] Homology comparisons can be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

[0048] % homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

[0049] Although this is a very simple and consistent method, it fails totake into consideration that, for example, in an otherwise identicalpair of sequences, one insertion or deletion will cause the followingamino acid residues to be put out of alignment, thus potentiallyresulting in a large reduction in % homology when a global alignment isperformed. Consequently, most sequence comparison methods are designedto produce optimal alignments that take into consideration possibleinsertions and deletions without penalising unduly the overall homologyscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximise local homology.

[0050] However, these more complex methods assign “gap penalties” toeach gap that occurs in th alignment so that, for the same number ofidentical amino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

[0051] Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7-58 to 7-60). However it is preferred to use the GCG Bestfit program. Anew tool, called BLAST 2 Sequences is also available for comparingprotein and nucleotide sequence (se FEMS Microbiol Lett 1999 174(2):247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov).

[0052] Although the final % homology can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix—the default matrixfor the BLAST suite of programs. GCG Wisconsin programs generally useeither the public default values or a custom symbol comparison table ifsupplied (see user manual for further details). It is preferred to usethe public default values for the GCG package, or in the case of othersoftware, the default matrix, such as BLOSUM62.

[0053] Once the software has produced an optimal alignment, it ispossible to calculate % homology, preferably % sequence identity. Thesoftware typically does this as part of the sequence comparison andgenerates a numerical result.

[0054] The terms “variant” or “derivative” in relation to the amino acidsequences of the present invention includes any substitution of,variation of, modification of, replacement of, deletion of or additionof one (or more) amino acids from or to the sequence providing theresultant amino acid sequence has a binding specificity, preferablyhaving at least the same binding specificity as the amino acid sequenceset out in SEQ ID No 1 or SEQ ID No 3 of the sequence listing herein.

[0055] SEQ ID No 1 or SEQ ID No 3 of the sequence listing herein may bemodified for use in the present invention. Typically, modifications aremade that maintain the binding specificity of the sequence. Amino acidsubstitutions may be made, for example from 1, 2 or 3 to 10 or 20substitutions provided that the modified sequence retains the requiredbinding specificity. Amino acid substitutions may include the use ofnon-naturally occurring analogues.

[0056] The 5T4 poypeptide of the present invention may also havedeletions, insertions or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalentmolecule. Deliberate amino acid substitutions may be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe binding specificity of the 5T4 poypeptide is retained. For example,negatively charged amino acids include aspartic acid and glutamic acid;positively charged amino acids include lysine and arginine; and aminoacids with uncharged polar head groups having similar hydrophilicityvalues include leucine, isoleucin, valine, glycine, alanine, asparagine,glutamine, serine, threonine, phenylalanine, and tyrosine.

[0057] Conservative substitutions may be made, for example according tothe Table below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar GAP ILV Polar - uncharged CSTM NQPolar - charged DE KR AROMATIC HFWY

[0058] Preferably, the 5T4 sequence is prepared by use of recombinanttechniques.

[0059] With regard to a fragment of the canine 5T4 sequence, preferablythe fragment conprises at least one, preferably some, most preferablyall of the amino acids 1-182 and/or 297-420 shown in SEQ ID No 1.

[0060] Nucleotide Sequences

[0061] It will be understood by a skilled person that numerous differentnucleotide sequences can encode the same 5T4 poypeptide of the presentinvention as a result of the degeneracy of the genetic code. Inaddition, it is to be understood that skilled persons may, using routinetechniques, make nucleotide substitutions that do not affect the 5T4poypeptide encoded by the nucleotide sequence of the present inventionto reflect the codon usage of any particular host organism in which the5T4 poypeptide of the present invention is to be expressed.

[0062] The terms “variant”, “homologue” or “derivative” in relation tothe nucleotide sequence set out in SEQ ID No 15 (see FIG. 26) of thepresent invention includes any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence providing the resultantnucleotide sequence codes for a canine or feline 5T4 polypeptide,preferably a polypeptide as set out in SEQ ID No 1 or 3 of the sequencelisting of the present invention.

[0063] As indicated above, with respect to sequence homology, preferablythere is at least 75%, more preferably at least 85%, more preferably atleast 90% homology to the sequences shown in the sequence listingherein. More preferably there is at least 95%, more preferably at least98%, homology. Nucleotide homology comparisons may be conducted asdescribed above. A preferred sequence comparison program is the GCGWisconsin Bestfit program described above. The default scoring matrixhas a match value of 10 for each identical nucleotide and −9 for eachmismatch. The default gap creation penalty is −50 and the default gapextension penalty is −3 for each nucleotide.

[0064] The present invention also encompasses nucleotide sequences thatare capable of hybridising selectively to the sequences presentedherein, or any variant, fragment or derivative thereof, or to thecomplement of any of the above. Nucleotide sequences are preferably atleast 15 nucleotides in length, more preferably at least 20, 30, 40 or50 nucleotides in length.

[0065] With regard to a fragment of the canine 5T4 sequence, preferablythe fragment conprises at least one, preferably some, most preferablyall of the nucleic acids 1-546 and/or 890-1263 shown in SEQ ID No 15.

[0066] Hybridisation

[0067] The term “hybridization” as used herein shall include “theprocess by which a strand of nucleic acid joins with a complementarystrand through base pairing”.

[0068] Nucleotide sequences of the invention capable of selectivelyhybridising to the nucleotide sequences presented herein, or to theircomplement, will be generally at least 75%, preferably at least 85 or90% and more preferably at least 95% or 98% homologous to thecorresponding nucleotide sequences presented herein over a region of atleast 20, preferably at least 25 or 30, for instance at least 40, 60 or100 or more contiguous nucleotides. Preferred nucleotide sequences ofthe invention will comprise regions homologous to the nucleotidesequence set out in SEQ ID No 2 or SEQ ID No 4 of the sequence listingsof the present invention preferably at least 80 or 90% and morepreferably at least 95% homologous to the nucleotide sequence set out inSEQ ID. NO 2 or 4 of the sequence listings of the present invention.

[0069] The term “selectively hybridizable” means that the nucleotidesequence used as a probe is used under conditions where a targetnucleotide sequence of the invention is found to hybridize to the probeat a level significantly above background. The background hybridizationmay occur because of other nucleotide sequences present, for example, inthe cDNA or genomic DNA library being screened. In this event,background implies a level of signal generated by interaction betweenthe probe and a non-specific DNA member of the library which is lessthan 10 fold, preferably less than 100 fold as intense as the specificinteraction observed with the target DNA. The intensity of interactionmay be measured, for example, by radiolabelling the probe, e.g. with³²P.

[0070] Hybridization conditions are based on the melting temperature(Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel(1987, Guide to Molecular Cloning. Techniques, Methods in Enzymology,Vol 152, Academic Press, San Diego Calif.), and confer a defined“stringency” as explained below.

[0071] Maximum stringency typically occurs at about Tm-5° C. (5° C.below the Tm of the probe); high stringency at about 5° C. to 10° C.below Tm; intermediate stringency at about 10° C. to 20° C. below Tm;and low stringency at about 20° C. to 25° C. below Tm. As will beunderstood by those of skill in the art, a maximum stringencyhybridization can be used to identify or detect identical nucleotidesequences while an intermediate (or low) stringency hybridization can beused to identify or detect similar or related polynucleotide sequences.

[0072] In a preferred aspect, the present invention covers nucleotidesequences that can hybridise to the nucleotide sequence of the presentinvention under stringent conditions (e.g. 65° C. and 0.1×SSC{1×SSC=0.15 M NaCl, 0.015 M Na₃ Citrate pH 7.0). Where the nucleotidesequence of the invention is double-stranded, both strands of theduplex, either individually or in combination, are encompassed by thepresent invention. Where the nucleotide sequence is single-stranded, itis to be understood that the complementary sequence of that nucleotidesequence is also included within the scope of the present invention.

[0073] Nucleotide sequences which are not 100% homologous to thesequences of the present invention but fall within the scope of theinvention can be obtained in a number of ways. Other variants of thesequences described herein may be obtained for example by probing DNAlibraries made from a range of sources. In addition, otherviral/bacterial or cellular homologues particularly cellular homologuesfound in mammalian cells (e.g. rat, mouse, bovine and primate cells),may be obtained and such homologues and fragments thereof in generalwill be capable of selectively hybridising to the sequences shown in thesequence listing herein. Such sequences may be obtained by probing cDNAlibraries made from or genomic DNA libraries from other animal species,and probing such libraries with probes comprising all or part of thenucleotide sequence set out in SEQ ID No 2 or SEQ ID No 4 of thesequence listings of the present invention under conditions of medium tohigh stringency. Similar considerations apply to obtaining specieshomologues and allelic variants of the amino acid and/or nucleotidesequences of the present invention.

[0074] Variants and strain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences within the sequences of the present invention. Conservedsequences can be predicted, for example, by aligning the amino acidsequences from several variants/homologues. Sequence alignments can beperformed using computer software known in the art. For example the GCGWisconsin PileUp program is widely used. The primers used in degeneratePCR will contain one or more degenerate positions and will be used atstringency conditions lower than those used for cloning sequences withsingle sequence primers against known sequences.

[0075] Alternatively, such nucleotide sequences may be obtained by sitedirected mutagenesis of characterised sequences, such as the nucleotidesequence set out in SEQ ID No 2 or SEQ ID No 4 of the sequence listingsof the present invention. This may be useful where for example silentcodon changes are required to sequences to optimise codon preferencesfor a particular host cell in which the nucleotide sequences are beingexpressed. Other sequence changes may be desired in order to introducerestriction enzyme recognition sites, or to alter the activity of the5T4 poypeptide encoded by the nucleotide sequences.

[0076] The nucleotide sequences of the present invention may be used toproduce a primer, e.g. a PCR primer, a primer for an alternativeamplification reaction, a probe e.g. labelled with a revealing label byconventional means using radioactive or non-radioactive labels, or thenucleotide sequences may be cloned into vectors. Such primers, probesand other fragments will be at least 15, preferably at least 20, forexample at least 25, 30 or 40 nucleotides in length, and are alsoencompassed by the term nucleotide sequence of the invention as usedherein.

[0077] The nucleotide sequences such as a DNA polynucleotides and probesaccording to the invention may be produced recombinantly, synthetically,or by any means available to those of skill in the art. They may also becloned by standard techniques.

[0078] In general, primers will be produced by synthetic means,involving a step wise manufacture of the desired nucleic acid sequenceone nucleotide at a time. Techniques for accomplishing this usingautomated techniques are readily available in the art.

[0079] Longer nucleotide sequences will generally be produced usingrecombinant means, for example using a PCR (polymerase chain reaction)cloning techniques. This will involve making a pair of primers (e.g. ofabout 15 to 30 nucleotides) flanking a region of the targeting sequencewhich it is desired to clone, bringing the primers into contact withmRNA or cDNA obtained from an animal or human cell, performing apolymerase chain reaction (PCR) under conditions which bring aboutamplification of the desired region, isolating the amplified fragment(e.g. by purifying the reaction mixture on an agarose gel) andrecovering the amplified DNA. The primers may be designed to containsuitable restriction enzyme recognition sites so that the amplified DNAcan be cloned into a suitable cloning vector

[0080] Due to the inherent degeneracy of the genetic code, other DNAsequences which encode substantially the same or a functionallyequivalent amino acid sequence, may be used to clone and express the 5T4poypeptide. As will be understood by those of skill in the art, it maybe advantageous to produce the 5T4 poypeptide—encoding nucleotidesequences possessing non-naturally occurring codons. Codons preferred bya particular prokaryotic or eukaryotic host (Murray E et al(1989) NucAcids Res 17:477-508) can be selected, for example, to increase the rateof the 5T4 poypeptide expression or to produce recombinant RNAtranscripts having desirable properties, such as a longer half-life,than transcripts produced from naturally occurring sequence.

[0081] Agent/Antibody

[0082] The fourth aspect of the invention provides an agent capable ofbinding specifically to canine or feline 5T4 protein. The agent may bean antibody.

[0083] WO 00/29428 describes antibodies which are capable of bindingspecifically to human 5T4. The present inventors have demonstrated thatthese antibodies do not cross-react with feline or canine 5T4.

[0084] As used herein, “antibody” includes a whole immunoglobulinmolecule or a part thereof or a bioisostere or a mimetic thereof or aderivative thereof or a combination thereof. Examples of a part thereofinclude: Fab, F(ab)′_(2,) and Fv. Examples of a bioisostere includesingle chain Fv (ScFv) fragments, chimeric antibodies, bifunctionalantibodies.

[0085] The term “mimetic” relates to any chemical which may be apeptide, polypeptide, antibody or other organic chemical which has thesame binding specificity as the antibody.

[0086] The term “derivative” as used herein includes chemicalmodification of an antibody. Illustrative of such modifications would bereplacement of hydrogen by an alkyl, acyl, or amino group.

[0087] A whole immunoglobulin molecule is divided into two regions:binding (Fab) domains that interact with the antigen and effector (Fc)domains that signal the initiation of processes such as phagocytosis.Each antibody molecule consists of two classes of polypeptide chains,light (L) chains and heavy (H) chains. A single antibody has twoindentical copies of the L chain and two of the H chain. The N-terminaldomain from each chain forms the variable regions, which constitute theantigen-binding sites. The C-terminal domain is called the constantregion. The variable domains of the H (V_(H)) and L (V_(L)) chainsconstitute an Fv unit and can interact closely to form a single chain Fv(ScFv) unit. In most H chains, a hinge region is found. This hingeregion is flexible and allows the Fab binding regions to move freelyrelative to the rest of the molecule. The hinge region is also the placeon the molecule most susceptible to the action of protease which cansplit the antibody into the antigen binding site (Fab) and the effector(Fc) region.

[0088] The domain structure of the antibody molecule is favourable toprotein engineering, facilitating the exchange between molecules offunctional domains carrying antigen-binding activities (Fabs and Fvs) oreffector functions (Fc). The structure of the antibody also makes iteasy to produce antibodies with an antigen recognition capacity joinedto molecules such as toxins, lymphocytes or growth factors.

[0089] Chimeric antibody technology amy involve the transplantation ofantibody variable domains from one species (for example, a mouse) ontoantibody constant domains from another species (for example a cat ordog).

[0090] Fab, Fv, and single chain Fv (ScFv) fragments with VH and VLjoined by a polypeptide linker exhibit specificities and affinities forantigen similiar to the original monoclonal antibodies. The ScFv fusionproteins can be produced with a non-antibody molecule attached to eitherthe amino or carboxy terminus. In these molecules, the Fv can be usedfor specific targeting of the attached molecule to a cell expressing theappropriate antigen. Bifunctional antibodies can also be created byengineering two different binding specificities into a single antibodychain. Bifunctional Fab, Fv and ScFv antibodies may comprise engineereddomains such as CDR grafted or humanised domains.

[0091] A large number of monoclonal antibodies and immunoglobulin-likemolecules are known which bind specifically to antigens present on thesurfaces of particular cell types. Procedures for identifying,characterising, cloning and engineering these molecules are wellestablished, for example using hybridomas derived from mice ortransgenic mice, phage-display libraries or scFv libraries. Genesencoding immunoglobulins or immunoglobulin-like molecules can beexpressed in a variety of heterologous expression systems. Largeglycosylated proteins including immunoglobulins are efficiently secretedand assembled from eukaryotic cells, particularly mammalian cells.Small, non-glycosylated fragments such as Fab, Fv, or scFv fragments canbe produced in functional form in mammalian cells or bacterial cells.

[0092] The antigen-binding domain may be comprised of the heavy andlight chains of an immunoglobulin, expressed from separate genes, or mayuse the light chain of an immunoglobulin and a truncated heavy chain toform a Fab or a F(ab)′₂ fragment. Alternatively, truncated forms of bothheavy and light chains may be used which assemble to form a Fv fragment.An engineered scFv fragment may also be used, in which case, only asingle gene is required to encode the antigen-binding domain.

[0093] In a preferred aspect, the present invention provides an ScFvantibody (ScFv Ab), capable of recognising canine 5T4 or feline 5T4.

[0094] The invention also provides a nucleotide sequence capable ofencoding such an antibody or derivative thereof and a vector comprisingsuch a nucleic acid sequence. The antibody or its precursor (i.e.nucleic acid encoding the antibody) may be used in a method to treatand/or prevent a disease.

[0095] For example, an ScFv Ab can be directly administered to a cat ordog either as a peptide (synthetically or genetically expressed) or as“naked DNA” (for example, in a plasmid) or via a delivery vehicle suchas a viral vector comprising the nucleotide sequence encoding the ScFvAb.

[0096] The antibody, or derivative thereof (for example anti-5T4 scFvs)may be used as an 5T4 targeting molecule. For example, they may be usedto (i) to target natural or exogenous 5T4 in situ and/or (ii) deliverimmune enhancer molecules, such as B7.1, to natural or exogenous 5T4 insitu (Carroll et al. (1998) J Natl Cancer Inst 90(24):1881-7). Thispotentiates the immunogenicity of 5T4 in the subject.

[0097] The term “binds specifically” as used herein is intended to meanthat the agent preferentially binds to canine and/or feline c5T4 than tohuman 5T4. Preferably the agent preferntailly binds to either canine orfeline 5T4 and does not bind or binds significantly less well to theother protein. If the agent is an antibody it may be raised againstcanine or feline 5T4 or one or more fragments thereof.

[0098] Vector Systems

[0099] The second aspect of the invention relates to a vector systemexpressing a polynucleotide encoding a canine or feline 5T4 antigen.

[0100] As used herein, a “vector system” may be any agent capable ofdelivering or maintaining nucleic acid in a host cell, and includesviral vectors, plasmids, naked nucleic acids, nucleic acids complexedwith polypeptide or other molecules and nucleic acids immobilised ontosolid phase particles. Such vectors are described in detail below. Itwill be understood that the present invention, in its broadest form, isnot limited to any specific vector for delivery of the 5T4-encodingnucleic acid.

[0101] The 5T4 antigen is “expressed” in accordance with the presentinvention by being produced in the cells of a host organism as a resultof translation, and optionally transcription, of the nucleic acidencoding the 5T4 antigen. Thus, 5T4 antigen is produced in situ in thecell. Since 5T4 is a transmembrane protein, the extracellular portionthereof is displayed on the surface of the cell in which it is produced.If necessary, therefore, the term “expression” includes the provision ofthe necessary signals to ensure correct processing of the 5T4 antigensuch that it is displayed on the cell surface and can interact with thehost immune system.

[0102] Vector

[0103] As it is well known in the art, a vector is a tool that allows orfaciliates the transfer of an entity from one environment to another. Inaccordance with the present invention, and by way of example, somevectors used in recombinant DNA techniques allow entities, such as asegment of DNA (such as a heterologous cDNA segment, such as aheterologous cDNA segment), to be transferred into a host and/or atarget cell for the purpose of replicating the vectors comprising thenucleotide sequences of the present invention and/or expressing theproteins of the invention encoded by the nucleotide sequences of thepresent invention. Examples of vectors used in recombinant DNAtechniques include but are not limited to plasmids, chromosomes,artificial chromosomes or viruses.

[0104] “Naked DNA”

[0105] The vectors comprising nucleotide sequences encoding 5T4poypeptide or 5T4-specific agent of the present invention may beadministered directly as “a naked nucleic acid construct”, preferablyfurther comprising flanking sequences homologous to the host cellgenome.

[0106] As used herein, the term “naked DNA” refers to a plasmidcomprising a nucleotide sequences encoding a 5T4 poypeptide or5T4-specific agent of the present invention together with a shortpromoter region to control its production. It is called “unaked” DNAbecause the plasmids are not carried in any delivery vehicle. When sucha DNA plasmid enters a host cell, such as a eukaryotic cell, theproteins it encodes are transcribed and translated within the cell.

[0107] Non-Viral Delivery

[0108] Alternatively, the vectors comprising nucleotide sequences of thepresent invention may be introduced into suitable host cells using avariety of non-viral techniques known in the art, such as transfection,transformation, electroporation and biolistic transformation.

[0109] As used herein, the term “transfection” refers to a process usinga non-viral vector to deliver a gene to a target mammalian cell.

[0110] Typical transfection methods include electroporation, DNAbiolistics, lipid-mediated transfection, compacted DNA-mediatedtransfection, liposomes, immunoliposomes, lipofectin, cationicagent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology1996 14; 556), multivalent cations such as spermine, cationic lipids orpolylysine, 1, 2-bis (oleoyloxy)-3-(trimethylammonio) propane(DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 NatureBiotechnology 16: 421) and combinations thereof.

[0111] Uptake of naked nucleic acid constructs by mammalian cells isenhanced by several known transfection techniques for example thoseincluding the use of transfection agents. Example of these agentsinclude cationic agents (for example calcium phosphate and DEAE-dextran)and lipofectants (for example lipofectam™ and transfectam™). Typically,nucleic acid constructs are mixed with the transfection agent to producea composition.

[0112] Viral Vectors

[0113] Alternatively, the vectors comprising nucleotide sequences of thepresent invention may be introduced into suitable host cells using avariety of viral techniques which are known in the art, such as forexample infection with recombinant viral vectors such as retroviruses,herpes simplex viruses and adenoviruses.

[0114] Preferably the vector is a recombinant viral vectors. Suitablerecombinant viral vectors include but are not limited to adenovirusvectors, adeno-associated viral (AAV) vectors, herpes-virus vectors, aretroviral vector, lentiviral vectors, baculoviral vectors, pox viralvectors or parvovirus vectors (see Kestler et al 1999 Human Gene Ther10(10):1619-32). In the case of viral vectors, gene delivery is mediatedby viral infection of a target cell.

[0115] The term “vector system” when applied to viral vector includes avector particle capable of infecting a mammalian cell. There is alsoprovided kits for the production of the vector particle, theconstituents of which will depend on the viral vector type on which thesystem is based. For example, a kit for a retrovirus may comprise:

[0116] i) a viral genome comprising a 5T4 nucleotide; and either

[0117] ii) one or more producer plasmids and a host cell, or

[0118] iii) a producer cell.

[0119] Retroviral Vectors

[0120] Examples of retroviruses include but are not limited to: murineleukemia virus (MLV), human immunodeficiency virus (HIV), equineinfectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Roussarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murineleukemia virus (Mo-MLV), FBR murine osteosarcoma virus. (FBR MSV),Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus(A-MLV); Avian myelocytomatosis virus-29 (MC29), and Avianerythroblastosis virus (AEV).

[0121] Preferred vectors for use in accordance with the presentinvention are recombinant viral vectors, in particular recombinantretroviral vectors (RRV) such as lentiviral vectors.

[0122] The term “recombinant retroviral vector” (RRV) refers to a vectorwith sufficient retroviral genetic information to allow packaging of anRNA genome, in the presence of packaging components, into a viralparticle capable of infecting a target cell. Infection of the targetcell includes reverse transcription and integration into the target cellgenome. The RRV carries non-viral coding sequences which are to bedelivered by the vector to the target cell. An RRV is incapable ofindependent replication to produce infectious retroviral particleswithin the final target cell. Usually the RRV lacks a functional gag-poland/or env gene and/or other genes essential for replication. The vectorof the present invention may be configured as a split-intron vector. Asplit intron vector is described in PCT patent application WO 99/15683.

[0123] A detailed list of retroviruses may be found in Coffin etal(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

[0124] Lentiviral Vectors

[0125] Lentiviruses can be divided into primate and non-primate groups.Examples of primate lentiviruses include but are not limited to: thehuman immunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiency virus(FIV) and bovine immunodeficiency virus (BIV).

[0126] A distinction between the lentivirus family and other types ofretroviruses is that lentivirus s have the capability to infect bothdividing and non-dividing cells (Lewis et al1992 EMBO. J 11: 3053-3058;Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect no-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

[0127] Adenoviruses

[0128] In one embodiment of the present invention, the features ofadenoviruses may be combined with the genetic stability ofretroviruses/lentiviruses which can be used to transduce target cells tobecome transient retroviral producer cells capable of stably infectneighbouring cells. Such retroviral producer cells which are engineeredto express a 5T4 poypeptide or 5T4-specific agent of the presentinvention can be implanted in organisms such as animals or humans foruse in the treatment of disease such as cancer.

[0129] Pox Viruses

[0130] Preferred vectors for use in accordance with the presentinvention are recombinant pox viral vectors such as fowl pox virus(FPV), entomopox virus, vaccinia virus such as NYVAC, canarypox virus,Modified vaccinia Ankara (MVA) or other non-replicating viral vectorsystems such as those described for example in WO 95/30018.

[0131] In a preferred embodiment the vector is MVA. General teachings onpox vectors and MVA can be found in WO 00/29428.

[0132] Expression of 5T4 proteins or antigens in recombinant poxviruses, such as vaccinia viruses, requires the ligation of vacciniapromoters to the nucleic acid encoding 5T4. In order to do this atransfer plasmid is constructed which contains at least one nucleic acidwhich codes for a 5T4 antigen flanked by MVA DNA sequences. When thistransfer plasmid is introduced into cells infected with MVA, homologousrecombination occurs causing the 5T4 nucleotide to be inserted into theMVA virus (Mackett et al 1982 PNAS 79: 7415-7419).

[0133] The transfer plasmid contains sequences flanking the left and theright side of a naturally occurring deletion, e.g. deletion II, withinthe MVA genome (Altenburger, W., Suter, C. P. and Altenburger J. (1989)Arch. Virol. 0.105, 15-27). The foreign DNA sequence is inserted betweenthe sequences flanking the naturally occurring deletion.

[0134] For the expression of at least one nucleic acid, it is necessaryfor regulatory sequences, which are required for the transcription ofthe nucleic acid to be present upstream of the nucleic acid. Suchregulatory sequences are known to those skilled in the art, and includesfor example those of the vaccinia 11 kDa gene as are described inEP-A-198,328, and those of the 7.5 kDa gene (EP-A-110,385).

[0135] The construct can be introduced into the MVA infected cells bytransfection, for example by means of calcium phosphate precipitation(Graham et al Virol. 52, 456-467-1973; Wigler et al Cell 777-785 [1979]by means of electroporation (Neumann et al EMBO J. 1, 841-845 [1982]),by microinjection (Graessmann et al Meth. Enzymology 101, 482-492(1983)), by means of liposomes (Straubinger et al Methods in Enzymology101, 512-527 (1983)), by means of spheroplasts (Schaffner, Proc. Natl.Acad. Sci. USA 77, 2163-2167 (1980)) or by other methods known to thoseskilled in the art. Transfection by means of liposomes is preferred.

[0136] Once the construct has been introduced into the eukaryotic celland the 5T4 antigen DNA has recombined with the viral DNA, the desiredrecombinant vaccinia virus, can be isolated, preferably with the aid ofa marker (Nakano et al Proc. Natl. Acad. Sci. USA. 79, 1593-1596 [1982],Franke et al Mol. Cell. Biol. 1918-1924 [1985], Chakrabarti et al Mol.Cell. Biol. 3403-3409 [1985], Fathi et al Virology 97-105 [1986]).

[0137] The present invention thus also provides a kit which comprises

[0138] a transfer plasmid which comprises a polynucleotide encoding a5T4 antigen flanked by MVA DNA sequences.

[0139] The kit may also include a cell infected with MVA, such as BHK-21or CEF cells, or wild-type MVA stock for infection purposes.

[0140] The nucleic acid must be inserted into a region (insertionregion) in the virus which does not affect virus viability of theresultant recombinant virus. Such regions can be readily identified in avirus by, for example, randomly testing segments of virus DNA forregions that allow recombinant formation without seriously affectingvirus viability of the recombinant. One region that can readily be usedand is present in many viruses is the thymidine kinase (TK) gene. Forexample, the TK gene has been found in all pox virus genomes examined[leporipoxvirus: Upton, et al J. Virology 60:920 (1986) (shope fibromavirus); capripoxvirus: Gershon, et al J. Gen. Virol. 70:525 (1989)(Kenya sheep-1); orthopoxvirus: Weir, et al J. Virol 46:530 (1983)(vaccinia); Esposito, et al Virology 135:561 (1984) (monkeypox andvariola virus); Hruby, et al PNAS, 80:3411 (1983) (vaccinia);Kilpatrick, et al Virology 143:399 (1985) (Yaba monkey tumour virus);avipoxvirus: Binns, et al J. Gen. Virol 69:1275 (1988) (fowlpox); Boyle,et al Virology 156:355 (1987) (fowlpox); Schnitzlein, et al J.Virological Method, 20:341 (1988) (fowlpox, quailpox); entomopox(Lytvyn, et al J. Gen. Virol 73:3235-3240 (1992)].

[0141] A promoter can be selected depending on the host and the target cII type. For example in poxviruses, pox viral promoters should be used,such as the vaccinia 7.5K, or 40K or fowlpox C1. Artificial constructscontaining appropriate pox sequences can also be used. Enhancer elementscan also be used in combination to increase the level of expression.Furthermore, the use of inducible promoters, which are also well knownin the art, are preferred in some embodiments. A particularly preferredpromoter is a modified H5 promoter (Wyatt et al (1996) Vaccine (4)1451-1458).

[0142] Hybrid Viral Vectors

[0143] In a further broad aspect, the present invention provides ahybrid viral vector system for in vivo delivery of a nucleotide sequenceencoding a 5T4 poypeptide or 5T4-specific agent of the presentinvention, which system comprises one or more primary viral vectorswhich encode a secondary viral vector, the primary vector or vectorscapable of infecting a first target cell and of expressing therein thesecondary viral vector, which secondary vector is capable of transducinga secondary target cell.

[0144] Preferably the primary vector is obtainable from or is based onan adenoviral vector and/or the secondary viral vector is obtainablefrom or is based on a retroviral vector preferably a lentiviral vector.

[0145] Targeted Vector

[0146] The term “targeted vector” refers to a vector whose ability toinfect/transfect/transduce a cell or to be expressed in a host and/ortarget cell is restricted to certain cell types within the hostorganism, usually cells having a common or similar phenotype.

[0147] Replication Vectors

[0148] The nucleotide sequences encoding the 5T4 poypeptide or5T4-specific agent of the present invention may be incorporated into arecombinant replicable vector. The vector may be used to replicate thenucleotide sequence in a compatible host cell. Thus in one embodiment ofthe present invention, the invention provides a method of making the 5T4poypeptide or 5T4-specific agent of the present invention by introducinga nucleotide sequence of the present invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell.

[0149] Expression Vector

[0150] Preferably, a nucleotide sequence of present invention which isinserted into a vector is operably linked to a control sequence that iscapable of providing for the expression of the coding sequence, such asthe coding sequence of the 5T4 poypeptide or 5T4-specific agent of thepresent invention by the host cell, i.e. the vector is an expressionvector. The 5T4 poypeptide or 5T4-specific agent produced by a hostrecombinant cell may be secreted or may be contained intracellularlydepending on the sequence and/or the vector used. As will be understoodby those of skill in the art, expression vectors containing the 5T4poypeptide or 5T4-specific agent coding sequences can be designed withsignal sequences which direct secretion of the 5T4 poypeptide or5T4-specific agent coding sequences through a particular prokaryotic oreukaryotic cell membrane.

[0151] Expression in Vitro

[0152] The vectors of the present invention may be transformed ortransfected into a suitable host cell and/or a target cell as describedbelow to provide for expression of a 5T4 poypeptide or 5T4-specificagent of the present invention. This process may comprise culturing ahost cell and/or target cell transformed with an expression vector underconditions to provide for expression by the vector of a coding sequenceencoding the 5T4 poypeptide or 5T4-specific agent and optionallyrecovering the expressed 5T4 poypeptide or 5T4-specific agent. Thevectors may be for example, plasmid or virus vectors provided with anorigin of replication, optionally a promoter for the expression of thesaid polynucleotide and optionally a regulator of the promoter. Thevectors may contain one or more selectable marker genes, for example, anampicillin resistance gene in the case of a bacterial plasmid or aneomycin resistance gene for a mammalian vector. The expression of the5T4 poypeptide or 5T4-specific agent of the invention may beconstitutive such that they are continually produced, or inducible,requiring a stimulus to initiate expression. In the case of inducibleexpression, 5T4 poypeptide or 5T4-specific agent production can beinitiated when require by, for example, addition of an inducer substanceto the culture medium, for example dexamethasone or IPTG.

[0153] Host/Target Cells

[0154] Host and/or target cells comprising nucleotide sequences of thepresent invention may be used to express the 5T4 polypeptide or5T4-specific agents of the present invention under in vitro, in vivo andex vivo conditions.

[0155] The term “host cell and/or target cell” includes any cellderivable from a suitable organism which a vector is capable oftransfecting or transducing. Examples of host and/or target cells caninclude but are not limited to cells capable of expressing the 5T4polypeptide or 5T4-specific agent of the present invention under invitro, in vivo and ex vivo conditions. Examples of such cells includebut are not limited to macrophages, endothelial cells or combinationsthereof. Further examples include respiratory airway epithelial cells,hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primarymammary epithelial cess and post-mitotically terminally differentiatednon-replicating cells such as macrophages and/or neurons.

[0156] In a preferred embodiment, the cell is a mammalian cell.

[0157] In a highly preferred embodiment, the cell is a canine or felinecell.

[0158] The term “organism” includes any suitable organism. In apreferred embodiment, the organism is a mammal. In a highly preferredembodiment, the organism is a dog or cat.

[0159] Although the 5T4 polypeptide or 5T4-specific agent of theinvention may be produced using prokaryotic cells as host cells, it ispreferred to use eukaryotic cells, for example yeast, insect ormammalian cells, in particular mammalian cells. Suitable host cellsinclude bacteria such as E. coli, yeast, mammalian cell lines and othereukaryotic cell lines, for example insect Sf9 cells.

[0160] The present invention also provides a method comprisingtransforming a host and/or target cell with a or the nucleotidesequence(s) of the present invention.

[0161] The term “transformed cell” means a host cell and/or a targetcell having a modified genetic structure. With the present invention, acell has a modified genetic structure when a vector according to thepresent invention has been introduced into the cell.

[0162] Host cells and/or a target cells may be cultured under suitableconditions which allow expression of the 5T4 polypeptide or 5T4-specificagent of the invention.

[0163] The present invention also provides a method comprising culturinga transformed host cell—which cell has been transformed with a or thenucleotide sequence(s) according to the present invention underconditions suitable for the expression of the 5T4 polypeptide or5T4-specific agent encoded by said nucleotide sequence(s).

[0164] The present invention also provides a method comprising culturinga transformed host cell—which cell has been transformed with a or thenucleotide sequence(s) according to the present invention or aderivative, homologue, variant or fragment thereof—under conditionssuitable for the expression of the 5T4 polypeptide or 5T4-specific agentencoded by said nucleotide sequence(s); and then recovering said 5T4polypeptide or 5T4-specific agent from the transformed host cellculture.

[0165] The 5T4 polypeptide or 5T4-specific agent of the presentinvention can be extracted from host cells by a variety of techniquesknown in the art, including enzymatic, chemical and/or osmotic lysis andphysical disruption. The 5T4 polypeptide or 5T4-specific agent may bepurified and isolated in a manner known per se.

[0166] Regulation of Expression In Vitro/Vivo/Ex Vivo

[0167] The present invention also encompasses gene therapy whereby the5T4 polypeptide or 5T4-specific agent encoding nucleotide sequence(s) ofthe present invention is regulated in vitrolin vivolex vivo. Forexample, expression regulation may be accomplished by administeringcompounds that bind to the 5T4 polypeptide or 5T4-specific agentencoding nucleotide sequence(s) of the present invention, or controlregions associated with the 5T4 polypeptide or 5T4-specific agentencoding nucleotide sequence of the present invention, or itscorresponding RNA transcript to modify the rate of transcription ortranslation.

[0168] Control Sequences

[0169] Control sequences operably linked to sequences encoding the 5T4polypeptide or 5T4-specific agent of the present invention includepromoters/enhancers and other expression regulation signals. Thesecontrol sequences may be selected to be compatible with the host celland/or target cell in which the expression vector is designed to beused. The control sequences may be modified, for example by the additionof further transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators.

[0170] Operably Linked

[0171] The term “operably linked” means that the components describedare in a relationship permitting them to function in their intendedmanner. A regulatory sequence “operably linked” to a coding sequence isligated in such a way that expression of the coding sequence is achievedunder condition compatible with the control sequences.

[0172] Preferably the nucleotide sequence of the present invention isoperably linked to a transcription unit.

[0173] The term “transcription unit(s)” as described herein are regionsof nucleic acid containing coding sequences and the signals forachieving expression of those coding sequences independently of anyother coding sequences. Thus, each transcription unit generallycomprises at least a promoter, an optional enhancer and apolyadenylation signal.

[0174] Promoters

[0175] The term promoter is well-known in the art and is used toindicate a transcription factor (complex) binding site. The termencompasses nucleic acid regions ranging in size and complexity fromminimal promoters to promoters including upstream elements andenhancers.

[0176] The promoter is typically selected from promoters which arefunctional in mammalian, cells, although prokaryotic promoters andpromoters functional in other eukaryotic cells may be used. The promoteris typically derived from promoter sequences of viral or eukaryoticgenes. For example, it may be a promoter derived from the genome of acell in which expression is to occur. With respect to eukaryoticpromoters, they may be promoters that function in a ubiquitous manner(such as promoters of α-actin, β-actin, tubulin) or, alternatively, atissue-specific manner (such as promoters of the genes for pyruvatekinase).

[0177] Hypoxic Promoters/Enhancers

[0178] The enhancer and/or promoter may be preferentially active in ahypoxic or ischaemic or low glucose environment, such that the 5T4polypeptide or 5T4-specific agent encoding nucleotide sequence(s) ispreferentially expressed in the particular tissues of interest, such asin the environment of a tumour, arthritic joint or other sites ofischaemia. Thus, any significant biological effect or deleterious effectof the 5T4 polypeptide-or 5T4-specific agent encoding nucleotidesequence(s) on the individual being treated may be reduced oreliminated. The enhancer element or other elements conferring regulatedexpression may be present in multiple copies. Likewise, or in addition,the enhancer and/or promoter may be preferentially active in one or morespecific cell types—such as any one or more of macrophages, endothelialcells or combinations thereof. Further examples may include but are notlimited to respiratory airway epithelial cells, hepatocytes, musclecells, cardiac myocytes, synoviocytes, primary mammary epithelial cellsand post-mitotically terminally differentiated non-replicating cellssuch as macrophages and/or neurons.

[0179] Tissue-Specific Promoters

[0180] The promoters of the present invention may be tissue-specificpromoters. Examples of suitable tissue restricted promoters/enhancersare those which are highly active in tumour cells such as apromoter/enhancer from a MUC1 gene, a CEA gene or a 5T4 antigen gene.Examples of temporally restricted promoters/enhancers are those whichare responsive to ischaemia and/or hypoxia, such as hypoxia responseelements or the promoter/enhancer of a grp78 or a grp94 gene. The alphafetoprotein (AFP) promoter is also a tumour-specific promoter. Onepreferred promoter-enhancer combination is a human cytomegalovirus(hCMV) major immediate early (MIE) promoter/enhancer combination.

[0181] Preferably the promoters of the present invention are tissuespecific. That is, they are capable of driving transcription of a 5T4polypeptide or 5T4-specific agent encoding nucleotide sequence(s) in onetissue while remaining largely “silent” in other tissue types.

[0182] The term “tissue specific” means a promoter which is notrestricted in activity to a single tissue type but which neverthelessshows selectivity in that they may be active in one group of tissues andless active or silent in another group. A desirable characteristic ofthe promoters of the present invention is that they posess a relativelylow activity in the absence of activated hypoxia-regulated enhancerelements, even in the target tissue. One means of achieving this is touse “silencer” elements which suppress the activity of a selectedpromoter in the absence of hypoxia.

[0183] The term “hypoxia” means a condition under which a particularorgan or tissue receives an inadequate supply of oxygen.

[0184] The level of expression of a or the 5T4 polypeptide or5T4-specific agent encoding nucleotide sequence(s) under the control ofa particular promoter may be modulated by manipulating the promoterregion. For example, different domains within a promoter region maypossess different gene regulatory activities. The roles of thesedifferent regions are typically assessed using vector constructs havingdifferent variants of the promoter with specific regions deleted (thatis, deletion analysis). This approach may be used to identify, forexample, the smallest region capable of conferring tissue specificity orthe smallest region conferring hypoxia sensitivity.

[0185] A number of tissue specific promoters, described above, may beparticularly advantageous in practising the present invention. In mostinstances, these promoters may be isolated as convenient restrictiondigestion fragments suitable for cloning in a selected vector.Alternatively, promoter fragments may be isolated using the polymerasechain reaction. Cloning of the amplified fragments may be facilitated byincorporating restriction sites at the 5′ end of the primers.

[0186] Inducible Promoters

[0187] The promoters of the present invention may also be promoters thatrespond to specific stimuli, for example promoters that bind steroidhormone receptors. Viral promoters may also be used, for example theMoloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter,the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus(CMV) IE promoter.

[0188] It may also be advantageous for the promoters to be inducible sothat the levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated.

[0189] Enhancer

[0190] In addition, any of these promoters may be modified by theaddition of further regulatory sequences, for example enhancersequences. Chimeric promoters may also be used comprising sequenceelements from two or more different promoters described above.

[0191] The term “enhancer” includes a DNA sequence which binds to otherprotein components of the transcription initiation complex and thusfacilitates the initiation of transcription directed by its associatedpromoter.

[0192] The in vitro/in vivo/ex vivo expression of the 5T4 polypeptide or5T4-specific agent of the present invention may be used in combinationwith a protein of interest (POI) or a nucleotide sequence of interest(NOI) encoding same.

[0193] Combination With POIs/NOIs

[0194] The 5T4 polypeptide or 5T4-specific agent of the presentinvention or nucleotide sequence encoding same may be used incombination with a POI, such as a pro-drug activating enzyme eitherdirectly or by vector delivery to, for example, a target cell or targettissue. Instead of or as well as being selectively expressed in targettissues, the 5T4 polypeptide or 5T4-specific agent of the presentinvention or nucleotide sequence encoding same may be used incombination with another POI such as a pro-drug activation enzyme orenzymes or with a nucleotide sequences of interest (NOI) or NOIs whichencode a pro-drug activation enzyme or enzymes. These pro-drugactivation enzyme or enzymes may have no significant effect or nodeleterious effect until the individual is treated with one or morepro-drugs upon which the appropriate pro-drug enzyme or enzymes act. Inthe presence of the active POI or NOI encoding same, treatment of anindividual with the appropriate pro-drug may lead to enhanced reductionin the disease condition such as a reduction in tumour growth orsurvival.

[0195] Pro-Drug POIs

[0196] A POI, such as a pro-drug activating enzyme, may be delivered toa disease site, such as a tumour site for the treatment of a cancer. Ineach case, a suitable pro-drug is used in the treatment of the patientin combination with the appropriate pro-drug activating enzyme. Anappropriate pro-drug may be administered in conjunction with the 5T4polypeptide or 5T4-specific agent or vector comprising the nucleotidesequence encoding same. Examples of pro-drugs include: etoposidephosphate (with alkaline phosphatase, Senter et al1988 Proc Natl AcadSci 85: 4842-4846); 5-fluorocytosine (with cytosine deaminase, Mullen etal1994 Cancer Res 54: 1503-1506);Doxorubicin-N-p-hydroxyphenoxyacetamide (with Penicillin-V-Amidase, Kerret al1990 Cancer Immunol Immunother 31: 202-206);Para-N-bis(2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidaseG2); Cephalosporin nitrogen mustard carbamates (with βb-lactamase);SR4233 (with P450 Reductase); Ganciclovir (with HSV thymidine kinase,Borrelli et all 988 Proc Natl Acad Sci 85: 7572-7576); mustard pro-drugswith nitroreductase (Friedlos et al1997 J Med. Chem 40: 1270-1275) andCyclophosphamide (with P450 Chen et al1996 Cancer Res 56: 1331-1340).

[0197] Examples of suitable pro-drug activation enzymes for use in theinvention include a thymidine phosphorylase which activates the5-fluoro-uracil pro-drugs capcetabine and furtulon; thymidine kinasefrom Herpes Simplex Virus which activates ganciclovir; a cytochrome P450which activates a pro-drug such as cyclophosphamide to a DNA damagingagent; and cytosine deaminase which activates 5-fluorocytosine.Preferably, a pro-drug activating enzyme of human origin is used.

[0198] POIs and NOIs

[0199] Other suitable proteins of interest (POIs) or NOIs encoding samefor use in the present invention include those that are of therapeuticand/or diagnostic application such as, but are not limited to: sequencesencoding cytokines, chemokines, hormones, antibodies, engineeredimmunoglobulin-like molecules, a single chain antibody, fusion proteins,enzymes, immune co-stimulatory molecules, immunomodulatory molecules,anti-sense RNA, a transdominant negative mutant of a target protein, atoxin, a conditional toxin, an antigen, a tumour suppressor protein andgrowth factors, membrane proteins, vasoactive proteins and peptides,anti-viral proteins and ribozymes, and derivatives therof (such as withan associated reporter group). When included, the POIs or NOIs encodingsame may be typically operatively linked to a suitable promoter, whichmay be a promoter driving expression of a ribozyme(s), or a differentpromoter or promoters, such as in one or more specific cell types.

[0200] Bystander Effect

[0201] The POI and/or NOI encoding same may be proteins which aresecreted from a cell. Alternatively the POI expression products are notsecreted and are active within the cell. In either event, it ispreferred for the POI expression product to demonstrate a bystandereffector or a distant bystander effect; that is the production of theexpression product in one cell leading to the killing of additional,related cells, either neighbouring or distant (e.g. metastatic), whichpossess a common phenotype.

[0202] Suitable POIs or NOIs encoding same for use in the presentinvention in the treatment or prophylaxis of cancer include proteinswhich: destroy the target cell (for example a ribosomal toxin), act as:tumour suppressors (such as wild-type p53); activators of anti-tumourimmune mechanisms (such as cytokines, co-stimulatory molecules andimmunoglobulins); inhibitors of angiogenesis; or which provide enhanceddrug sensitivity (such as pro-drug activation enzymes); indirectlystimulate destruction of target cell by natural effector cells (forexample, strong antigen to stimulate the immune system or convert aprecursor substance to a toxic substance which destroys the target cell(for example a prodrug activating enzyme). Encoded proteins could alsodestroy bystander tumour cells (for example with secreted antitumourantibody-ribosomal toxin fusion protein), indirectly stimulatedestruction of bystander tumour cells (for example cytokines tostimulate the immune system or procoagulant proteins causing localvascular occlusion) or convert a precursor substance to a toxicsubstance which destroys bystander tumour cells (eg an enzyme whichactivates a prodrug to a diffusible drug).

[0203] Also, the delivery of NOI(s) encoding antisense transcripts orribozymes which interfere with expression of cellular genes for tumourpersistence (for example against aberrant myc transcripts in Burkittslymphoma or against bcr-abl transcripts in chronic myeloid leukemia. Theuse of combinations of such POIs and/or NOIs encoding same is alsoenvisaged.

[0204] Examples of hypoxia regulatable therapeutic NOIs can be found inPCT/GB95/00322 (WO-A9521927).

[0205] Coupling

[0206] The 5T4 polypeptide or 5T4-specific agent of the presentinvention can be coupled to other molecules using standard methods. Theamino and carboxyl termini of 5T4 polypeptide or 5T4-specific agent maybe isotopically and nonisotopically labeled with many techniques, forexample radiolabeling using conventional techniques (tyrosineresidues-chloramine T, iodogen, lactoperoxidase; lysineresidues-Bolton-Hunter reagent). These coupling techniques are wellknown to those skilled in the art. The coupling technique is chosen onthe basis of the functional groups available on the amino acidsincluding, but not limited to amino, sulfhydral, carboxyl, amide,phenol, and imidazole. Various reagents used to effect these couplingsinclude among others, glutaraldehyde, diazotized benzidine,carbodiimide, and p-benzoquinone.

[0207] Chemical Coupling

[0208] The 5T4 polypeptide or 5T4-specific agent of the presentinvention may be chemically coupled to isotopes, enzymes, carrierproteins, cytotoxic agents, fluorescent molecules, radioactivenucleotides and other compounds for a variety of applications includingbut not limited to imaging/prognosis, diagnosis and/or therapy. Theefficiency of the coupling reaction is determined using differenttechniques appropriate for the specific r action. For example,radiolabeling of an 5T4 polypeptide or 5T4-specific agent peptide with¹²⁵I is accomplished using chloramine T and Na¹²⁵I of high specificactivity. The reaction is terminated with sodium metabisulfite and themixture is desalted on disposable columns. The labeled peptide is elutedfrom the column and fractions are collected. Aliquots are removed fromeach fraction and radioactivity measured in a gamma counter. In thismanner, the unreacted Na ¹²⁵ I is separated from the labeled 5T4polypeptide or 5T4-specific agent. The peptide fractions with thehighest specific radioactivity are stored for subsequent use such asanalysis of the ability to bind to a 5T4 polypeptide or 5T4-specificagent.

[0209] Imaging

[0210] The use of labelled 5T4-specific agents of the present inventionwith short lived isotopes enables visualization quantitation of 5T4 invivo using autoradiographic, or modem radiographic or other membranebinding techniques such as positron emission tomography in order tolocate tumours with 5T4. This application provides important diagnosticand/or prognostic research tools.

[0211] Conjugates

[0212] In other embodiments, the 5T4 polypeptide or 5T4-specific agentof the invention is coupled to a scintigraphic radiolabel, a cytotoxiccompound or radioisotope, an enzyme for converting a non-toxic prodruginto a cytotoxic drug, a compound for activating the immune system inorder to target the resulting conjugate to a disease site such as acolon tumour, or a cell-stimulating compound. Such conjugates have a“binding portion”, which consists of the 5T4 polypeptide or 5T4-specificagent of the invention, and a “functional portion”, which consists ofthe radiolabel, toxin or enzyme. Different 5T4 polypeptide or5T4-specific agents can be synthesized for use in several applicationsincluding but not limited to the linkage of a 5T4 polypeptide or5T4-specific agent to cytotoxic agents for targeted killing of cellsthat bind the 5T4 polypeptide or 5T4-specific agent.

[0213] The binding portion and the functional portion of the conjugate(if also a peptide or poypeptide) may be linked together by any of theconventional ways of cross linking polypeptides, such as those generallydescribed in O'Sullivan et al(Anal. Biochem 1979: 100, 100-108). Forexample, one portion may be enriched with thiol groups and the otherportion reacted with a bifunctional agent capable of reacting with thosethiol groups, for example the N-hydroxysuccinimide ester of iodoaceticacid (NHIA) or N-succinimidyl-3-(2-pyridyidithio)propionate (SPDP).Amide and thioether bonds, for example achieved withm-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stablein vivo than disulphide bonds.

[0214] Alternatively, if the binding portion contains carbohydrates,such as would be the case for an antibody or some antibody fragments,the functional portion may be linked via the carbohydrate portion usingthe linking technology in EP 0 088 695.

[0215] The functional portion of the conjugate may be an enzyme forconverting a non-toxic prodrug into a toxic drug, for example theconjugates of Bagshawe and his colleagues (Bagshawe (1987) Br. J. Cancer56, 531; Bagshawe et al(Br. J. Cancer 1988: 58, 700); WO 88/07378) orcyanide-releasing systems (WO 91/11201).

[0216] The conjugate may be purified by size exclusion or affinitychromatography, and tested for dual biological activities. The antigenimmunoreactivity may be measured using an enzyme-linked immunosorbentassay (ELISA) with immobilised antigen and in a live cellradioimmunoassay. An enzyme assay may be used for β-glucosidase using asubstrate which changes in absorbance when the glucose residues arehydrolysed, such as oNPG (o-nitrophenyl-βD-glucopyranoside), liberating2-nitrophenol which is measured spectrophotometrically at 405 nm.

[0217] The stability of the conjugate may be tested in vitro initiallyby incubating at 37° C. in serum, followed by size exclusion FPLCanalysis. Stability in vivo can be tested in the same way in mice byanalysing the serum at various times after injection of the conjugate.In addition, it is possible to radiolabel the 5T4 polypeptide or5T4-specific agent with ¹²⁵I, and the enzyme with ¹³¹I beforeconjugation, and to determine the biodistribution of the conjugate, free5T4 polypeptide or 5T4-specific agent and free enzyme in mice.

[0218] Alternatively, the conjugate may be produced as a fusion compoundby recombinant DNA techniques whereby a length of DNA comprisesrespective regions encoding the two portions of the conjugate eitheradjacent to one another or separated by a region encoding a linkerpeptide which does not destroy the desired properties of the conjugate.

[0219] Conceivably, two of the functional portions of the compound mayoverlap wholly or partly. The DNA is then expressed in a suitable hostin known ways.

[0220] Diagnostic Kits

[0221] The present invention also includes diagnostic methods and kitsfor detection and measurement of 5T4 in biological fluids and tissues,and for localization of 5T4 in tissues. The 5T4 polypeptide or5T4-specific agent of thepresent invention that possess high bindingspecificity can be used to establish easy to use kits for rapid,reliable, sensitive, and specific measurement and localization of a 5T4in extracts of plasma, urine, tissues, and in cell culture media. The5T4 polypeptide or 5T4-specific agent of the present invention may alsobe used in a diagnostic method and kit to permit detection ofcirculating 5T4 which, in certain situations, may indicate theprogression of a disease state such as the spread of micrometastases byprimary tumours in situ.

[0222] These kits may include but are not limited to the followingtechniques; competitive and non-competitive assays, radioimmunoassay,bioluminescence and chemiluminescence assays, fluorometric assays,sandwich assays, immunoradiometric assays, dot blots, enzyme linkedassays including ELISA, microtiter plates, antibody coated strips ordipsticks for rapid monitoring of urine or blood, andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished. Intraassay and interassay variation is established at 20%,50% and 80% points on the standard curves of displacement or activity.

[0223] One example of an assay kit commonly used in research and in theclinic is a radioimmunoassay (RIA) kit. After successful radioiodinationand purification of a 0.5T4-specific agent, the antiserum possessing thehighest titer is added at several dilutions to tubes containing arelatively constant amount of radioactivity, such as 10,000 cpm, in asuitable buffer system. Other tubes contain buffer or preimmune serum todetermine the non-specific binding. After incubation at 4° C. for 24hours, protein A is added and the tubes are vortexed, incubated at roomtemperature for 90 minutes, and centrifuged at approximately 2000-2500times g at 4° C. to precipitate the complexes of antiserum bound to thelabeled 5T4-specific antibody. The supernatant is removed by aspirationand the radioactivity in the pellets counted in a gamma counter. Theantiserum dilution that binds approximately 1.0 to 40% of the labeled5T4-specific agent after subtraction of the non-specific binding isfurther characterized.

[0224] Immunohistochemistry

[0225] An immunohistochemistry kit may also be used for localization of5T4 in tissues and cells. This immunohistochemistry kit providesinstructions, a 5T4-specific antibody, and possibly blocking serum andsecondary antiserum linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art. This immunohistochemistry kit permits localizationof 5T4 in tissue sections and cultured cells using both light andelectron microscopy. It is used for both research and clinical purposes.For example, tumours are biopsied or collected and tissue sections cutwith a microtome to examine sites of 5T4 production. Such information isuseful for diagnostic and possibly therapeutic purposes in the detectionand treatment of diseases such as cancer.

[0226] Foetal Cell Analysis

[0227] The 5T4 polypeptide and 5T4-specific agents of the presentinvention are also useful in methods for isolating foetal cells frommaternal blood. Isolation of foetal cells from maternal blood has beenproposed as a non-invasive alternative to aminocentesis (see WO97/30354).

[0228] 5T4 is known to be expressed at very high levels on trophoblasts.Thus an antibody against 5T4 may be used to isolate trophoblasts frommaternal blood.

[0229] Thus the present invention also provides a method for isolating afoetal cell from maternal blood using an 5T4-specific agent Of thepresent invention

[0230] The foetal cell may, for example, be a trophoblast or anerythrocyte.

[0231] The maternal/foetal cells are preferably from a cat or a dog,such that the isolation method is part of a veterinary application.

[0232] The isolation process may form part of a diagnostic method. Forexample, the foetal cells may then be subject to biochemical or geneticsampling. Such a procedure sould be used to test for foetalabnormalities such as Downs syndrome, or to determine the sex of thefoetus(es).

[0233] Combination Therapy

[0234] The 5T4 polypeptide or 5T4-specific agents of the presentinvention may be used in combination with other compositions andprocedures for the treatment of diseases. By way of example, the 5T4polypeptide or 5T4-specific agents may also be used in combination withconventional treatments of diseases such as cancer. By ways of furtherexample, a tumor may be treated conventionally with surgery, radiationor chemotherapy combined with a 5T4 polypeptide or 5T4-specific agent ora 5T4 polypeptide or 5T4-specific agent may be subsequently administeredto the patient to extend the dormancy of micrometastases and tostabilize any residual primary tumor.

[0235] Delivery

[0236] The 5T4 polypeptide or 5T4-specific agent can be delivered withanother therapeutically effective agent at the same moment in time andat the same site. Alternatively, the 5T4 polypeptide or 5T4-specificagent and the therapeutically effective agent may be delivered at adifferent time and to a different site. The 5T4 polypeptide or5T4-specific agent and the therapeutically effective agent may even bedelivered in the same delivery vehicle for the prevention and/ortreatment of a disease condition such as cancer.

[0237] Therapeutic strategies based on the use of the 5T4-specific agentinclude the recruitment and activation of T cells by using a fusion a5T4-specific agent fragment with the bacterial superantigenstaphylococcal enterotoxin (Dohlsten et al1994) or by using bispecificantibodies, directed to both 5T4 and the T-cell CD3 antigen (Kroesen etal 1994). Anti-5T4 antibodies may also be conjugated to differentbacterial toxins to yield potent immunotoxins (LeMaistre et al 1987;Zimmermann et al 1997).

[0238] 5T4 polypeptide or 5T4-specific agents may be used in combinationwith cytotoxic agents for the prevention and/or treatment of diseasestates such as angiogenesis and/or cancer. Cytotoxic agents such asricin, linked to a 5T4-specific agent may provide a tool for thedestruction of cells expressing 5T4. These cells may be found in manylocations, including but not limited to, micrometastases and primarytumours.

[0239] Dosage

[0240] The dosage of the composition of the present invention willdepend on the disease state or condition being treated and otherclinical factors such as weight and condition of the human or animal andthe route of administration of the compound. Depending upon thehalf-life of the active agent in the particular animal or human, theagent (e.g. anti-5T4 antibody) can be administered between several timesper day to once a week. It is to be understood that the presentinvention has application primarily for veterinary use. The methods ofthe present invention contemplate single as well as multipleadministrations, given either simultaneously or over an extended periodof time.

[0241] Formulations

[0242] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0243] The compositions of the present invention may be effective inpreventing and/or treating diseases such as cancer related diseases. Thepresent invention includes the method of treating diseases such ascancer related disease with an effective amount of a composition of thepresent invention. The 5T4 polypeptide or 5T4-specific agent of thepresent invention can be provided as a synthetic peptide or an isolatedand substantially purified proteins or protein fragments or acombination thereof in pharmaceutically acceptable compositions usingformulation methods known to those of ordinary skill in the art. Thesecompositions can be administered by standard routes. These include butare not limited to: oral, rectal, ophthalmic (including intravitreal orintracameral), nasal, topical (including buccal and sublingual),intrauterine, vaginal or parenteral (including subcutaneous,intraperitoneal, intramuscular, intravenous, intradermal, intracranial,intratracheal, and epidural) transdermal, intraperitoneal, intracranial,intracerebroventricular, intracerebral, intravaginal, intrauterine, orparenteral (e.g., intravenous, intraspinal, subcutaneous orintramuscular) routes.

[0244] The compositions may conveniently be presented in unit dosageform and may be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

[0245] In addition, the compositions of the present invention may beincorporated into biodegradable polymers allowing for sustained releaseof the compound, the polymers being implanted in the vicinity of wheredrug delivery is desired, for example, at the site of a tumor orimplanted so that the active agent is slowly released systemically. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al(J. Neurosurg 1991 74:441-446). Osmotic minipumpsmay also be used to provide controlled delivery of high concentrationsof active agents through cannulae to the site of interest, such asdirectly into a metastatic growth or into the vascular supply to thattumor.

[0246] The 5T4 polypeptide or 5T4-specific agents of the presentinvention may be linked to cytotoxic agents which are infused in amanner designed to maximize delivery to the desired location. Forexample, ricin-linked high affinity 5T4 polypeptide or 5T4-specificagents are delivered through a cannula into vessels supplying the targetsite or directly into the target. Such agents are also delivered in acontrolled manner through osmotic pumps coupled to infusion cannulae.

[0247] Preferred unit dosage formulations are those containing a dailydose or unit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient. It should beunderstood that in addition'to the ingredients, particularly mentionedabove, the formulations of the present invention may include otheragents conventional in the art having regard to the type of formulationin question.

[0248] The conjugates may be administered in any suitable way, usuallyparenterally, for example intravenously or intraperitoneally, instandard sterile, non-pyrogenic formulations of diluents and carriers,for example isotonic saline (when administered intravenously). Once thconjugate has bound to the target cells and been cleared from thebloodstream (if necessary), which typically takes a day or so, thepro-drug is administered, usually as a single infused dose, or thetumour is imaged. If needed, because the conjugate may be immunogenic,cyclosporin or some other immunosuppressant can be administered toprovide a longer period for treatment but usually this will not benecessary.

[0249] The timing between administrations of the conjugate and pro-drugmay be optimised in a routine way since disease/normal tissue ratios ofconjugate (at least following intravenous delivery) are highest afterabout 4-6 days, whereas at this time the absolute amount of conjugatebound to the 5T4, in terms of percent of injected dose per gram, islower than at earlier times.

[0250] Therefore, the optimum interval between administration of theconjugate and the pro-drug will be a compromise between peakconcentration of the enzyme at the disease site and the bestdistribution ratio between disease and normal tissues. The dosage of theconjugate will be chosen by the physician according to the usualcriteria. At least in the case of methods employing a targeted enzymesuch as β-glucosidase and intravenous amygdalin as the toxic pro-drug, 1to 50 daily doses of 0.1 to 10.0 grams per square metr of body surfacearea, preferably 1.0-5.0 g/m² are likely to be appropriate. For oraltherapy, three doses per day of 0.05 to 10.0 g, preferably 1.0-5.0 g,for one to fifty days may be appropriate. The dosage of the conjugatewill similarly be chosen according to normal criteria, particularly withreference to the type, stage and location of the disease tissue and theweight of the patient. The duration of treatment will depend in partupon the rapidity and extent of any immune reaction to the conjugate.

[0251] The functional portion of the conjugate, when used for diagnosis,usually comprises and may consist of a radioactive atom forscintigraphic studies, for example technetium 99 m (^(99m)Tc) oriodine-123 (¹²³I), or a spin label for nuclear magnetic resonance (nmr)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-313, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0252] When used in a compound for selective destruction of, forexample, the tumour, the functional portion of the 5T4-specific agentmay comprise a highly radioactive atom, such as iodine-131, rhenium-186,rhenium-188, yttrium-90 or lead-212, which emits enough energy todestroy neighbouring cells, or a cytotoxic chemical compound such asmethotrexate, adriamicin, vinca alkaliods (vincristine, vinblastine,etoposide), daunorubicin or other intercalating agents.

[0253] The radio- or other labels may be incorporated in the5T4-specific agent conjugate in known ways. For example, the peptide maybe biosynthesised or may be synthesised by chemical amino acid synthesisusing suitable amino acid precursors involving, for example, fluorine-19in place of hydrogen. Labels such as ^(99m)Tc, ¹²³I, ¹⁸⁶Rh, ¹⁸⁸Rh and¹¹¹In can be attached via a cysteine residue in the peptide. Yttrium-90can be attached via a lysine residue. The IODOGEN method (Fraker etal(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used toincorporate iodine-123. “Monoclonal Antibodies in Immunoscinigraphy”(Chatal, CRC Press 1989) describes other methods in detail.

[0254] Pharmaceutical Compositions

[0255] The third aspect of the invention refers to vaccines priming andboosting compositions, agents and kits. Any and all of the products ofthis aspect of the invention can be considered to be a pharmaceuticalcomposition.

[0256] The pharmaceutical compositions will typically be for animalusage in veterinary medicine and will typically comprise any one or moreof a pharmaceutically acceptable diluent, carrier, or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington's.Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

[0257] Preservatives, stabilizers, dyes and even flavouring agents maybe provided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

[0258] There may be different composition/formulation requirementsdependent on the different delivery systems. By way of example, thepharmaceutical composition of the present invention may be formulated tobe delivered using a mini-pump or by a mucosal route, for example, as anasal spray or aerosol for inhalation or ingestable solution, orparenterally in which the composition is formulated by an injectableform, for delivery, by, for example, an intravenous, intramuscular orsubcutaneous route. Alternatively, the formulation may be designed to bedelivered by both routes.

[0259] Where the pharmaceutical composition is to be delivered mucosallythrough the gastrointestinal mucosa, it should be able to remain stableduring transit though the gastrointestinal tract; for example, it shouldbe resistant to proteolytic degradation, stable at acid pH and resistantto the detergent effects of bile.

[0260] Where appropriate, the pharmaceutical compositions can beadministered by inhalation, in the form of a suppository or pessary,topically in the form of a lotion, solution, cream, ointment or dustingpowder, by use of a skin patch, orally in the form of tablets containingexcipients such as starch or lactose or chalk, or in capsules or ovuleseither alone or in admixture with excipients, or in the form of elixirs,solutions or suspensions containing flavouring or colouring agents, orthey can be injected parenterally, for example intravenously,intramuscularly or subcutaneously. For parenteral administration, thecompositions may be best used in the form of a sterile aqueous solutionwhich may contain other substances, for example enough salts ormonosaccharides to make the solution isotonic with blood. For buccal orsublingual administration the compositions may be administered in theform of tablets or lozenges which can be formulated in a conventionalmanner.

[0261] Administration

[0262] Typically, a physician will determine the actual dosage whichwill be most suitable for an individual subject and it will vary withthe age, weight and response of the particular patient and severity ofthe condition. The dosages below are exemplary of the average case.There can, of course, be individual instances where higher or lowerdosage ranges are merited.

[0263] The compositions (or component parts thereof) of the presentinvention may be administered orally. In addition or in the alternativethe compositions (or component parts thereof) of the present inventionmay be administered by direct injection. In addition or in thealternative the compositions (or component parts thereof) of the presentinvention may be administered topically. In addition or in thealternative the compositions (or component parts thereof) of the presentinvention may be administered by inhalation. In addition or in thealternative the compositions (or component parts thereof) of the presentinvention may also be administered by one or more of: parenteral,mucosal, intramuscular, intravenous, subcutaneous, intraocular ortransdermal administration means, and are formulated for suchadministration.

[0264] By way of further example, the pharmaceutical composition of thepresent invention may be administered in accordance with a regimen of 1to 10 times per day, such as once or twice per day. The specific doselevel and frequency of dosage for any particular patient may be variedand will depend upon a variety of factors including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the age, body weight, general health, sex, diet, modeand time of administration, rate of excretion, drug combination, theseverity of the particular condition, and the host undergoing therapy.

[0265] The term “administered” also includes but is not limited todelivery by a mucosal route, for example, as a nasal spray or aerosolfor inhalation or as an ingestable solution; a parenteral route wheredelivery is by an injectable form, such as, for example, an intravenous,intramuscular or subcutaneous route.

[0266] Hence, the pharmaceutical composition of the present inventionmay be administered by one or more of the following routes: oraladministration, injection (such as direct injection), topical,inhalation, parenteral administration, mucosal administration,intramuscular administration, intravenous administration, subcutaneousadministration, intraocular administration or transdermaladministration.

[0267] Diseases

[0268] The present invention provides a method for treating and/orpreventing a disease in a subject.

[0269] The subject may be an animal, preferably a companion animal, mostpreferably a cat or a dog. Preferably the subject is a dog.

[0270] The compositions of the invention are contemplated to exhibittherapeutic and/or prophylactic activity, for example, in the treatmentand/or prophylaxis of tumours or other diseases associated with cellproliferation, infections and inflammatory conditions. The compositionsare particularly effective for cancer immunotherapy.

[0271] In particular the compositions are effective for immunotherapy ofthe feline cancers shown in Table 1 and the canine cancers shown inTable 2. In a highly preferred embodiment there is provided a method fortreating and/or preventing a mammary tumour in a dog.

[0272] In general the pharmaceutical compositions of the invention maybe used in the treatment of disorders such as those listed inWOA-98/09985.

[0273] The invention is further described, for the purposes ofillustration only, in the following examples in which reference is madeto the following Figures.

FIGURES

[0274]FIG. 1 shows human and canine placental sections stained with Y1an anti-5T4 antibody. A) Human placenta stained with Y1, b) Canineplacenta negative control, c) canine placenta stained with Y1

[0275]FIG. 2 shows western blotting of truncated human and canine 5T4.

[0276]FIG. 3 shows a comparison of feline, canine, human and murineamino acid sequences.

[0277]FIG. 4 shows 293T cells transiently transfected with pIRESneo (A)or pIRES c5T4 (b), formalin fixed and stained with anti-5T4 peptideantiserum.

[0278]FIG. 5 shows western blotting of cell supernatants to demonstratethe presence of a pIRES_c5T4 TM-stable transfectant.

[0279]FIG. 6 shows silver staining of c5T4TM-EKMycHis.

[0280] 1. MW marker

[0281] 2. Purified c5T4TM-EKMycHis (1.2 μg for silver stain or 675 ngfor western blots

[0282] 3. MVA-c5T4LacZ-infected CEF cell lysate (cells infected at MOIof 1 and incubated for 24 h)

[0283]FIG. 7 shows western blotting a) anti-c-myc, b) Y1.

[0284]FIG. 8 demonstrates the ability of both canine and feline cells tosupport expression of 5T4 from recombinant MVA.

[0285] 1 CF2TH (canine thymus)

[0286] 2 D17(poodle osteosarcoma)

[0287] 3 AKD (foetal feline lung)

[0288] 4 CRFK (feline kidney)

[0289] 5 293T (human kidney)

[0290]FIG. 9 illustrates cloning c5T4 into the MVA transfer vectors

[0291]FIG. 10 shows CEFs uninfected (panels A and C) and infected withMVA-c5T41lacZ (panels B and D) and immunostained with Y1 (Panels A andB) or Y3-P3 (Panels C and D).

[0292]FIG. 11 shows western blotting of lysates from CEFs infected withMVA-c5T4lacZ CEF only

[0293] 2: MVA_h5T4 (reduced)

[0294] 3: MVA_h5T4 (unreduced)

[0295] 4: MVA (wt)

[0296] 5: MVA-c5T4lacZ (reduced)

[0297] 6: MVA-c5T4lacZ (unreduced).

[0298]FIG. 12 shows expression of c5T4 in canine thymic cells.

[0299] 1 CF2TH-MVA-h5T4

[0300] 2 CF2TH-MVA-c5T4lacZ

[0301] 3 CF2TH-MVA (wt)

[0302]FIG. 13 shows examples of 5T4 positive canine tumour samples. A)mammary carcinoma, B) anal aprocrine carcinoma

EXAMPLES Example 1 Production of Production of new anti-5T4 antibodies

[0303] Antibody preparations were raised in chickens against a pool ofthree, 20 amino acid, 5T4 peptides. Regions that are likely to besurface exposed (hydrophilic), flexible and charged are good candidatesfor immunogenic peptides. The peptides chosen were as follows:-

[0304] Pep1

[0305] CRYEINADPRLTNLSSNSDV

[0306] Pep2

[0307] CLNHIVPPEDERQNRSFEG

[0308] Pep3

[0309] NLSGSRLDEVRAGAFEHLPSLRC

[0310] One such antiserum, Y1, was demonstrated to recognise both humanand canine 5T4, expressed on placenta (FIG. 1)

[0311] Y1 antibody preparation was used for most of the analysis ofcanine and feline tissue samples and in the analysis of 5T4 expressingcell lines and the recombinant MVA.

[0312] A concern with Y1 was higher than desired background staining. Inan attempt to reduce this, affinity purification was carried out usingindividual columns of each of the 3 original peptides used in theimmunisations. It was demonstrated that the majority of antigenicactivity was directed against peptide 3 and so this was used for furtherpurification. The antibody preparation Y3 was successfully purified andsignificantly improved (see below) using this approach.

[0313] Western blotting of Myc-His tagged, truncated human and canine5T4 demonstrates that peptide 3 purified Y3 antiserum (Y3-P3) resultedin a high activity anti-5T4 antiserum with low background (FIG. 2).(Conversely, purification of Y1 by this method appeared to result in aloss of activity).

Example 2 Isolation of a canine and feline 5T4 genes

[0314] A canine genomic library in λ dash was obtained from Stratageneand screened according to manufacturers instructions using aradiolabelled probe derived from the human 5T4 cDNA. A number of cloneswere Isolated and purified to homogeneity. The gene was identified bysouthern blot, subcloned into pBSII and sequenced. The canine gene wasthen used to probe a feline genomic library in the same way and a clonewas isolated, subcloned and sequence. A comparison of the feline,canine, human and murine amino acid sequences is shown in FIG. 3.

[0315] Cloning into Expression Vectors

[0316] The full length c5T4 coding region was amplified by PCR from apBSII subclone and cloned into the pIRES neo expression vector(Clontech)

[0317] 293T cells were transfected with pIRES_c5T4, pIRES_h5T4 and pIRESneo. Transfected cells were immunostained with the H8 mAb and the anti5T4 peptide antiserum, Y1. The Y1 was able to recognise both human andcanine 5T4. Transfected cells were also formalin fixed (see FIG. 4).

Example 3 Cloning of Truncated c5T4 With Optimised Kozac Sequence

[0318] To produce the c5T4 protein in sufficient quantities fordownstream applications such as ELISA, a truncated version of the gene,lacking the transmembrane region and cytoplasmic tail, but taggedc-terminally with a c-myc epitope and 6 histidines (Myc-His), wasconstructed by PCR to give a secreted protein that could be readilypurified from Chinese Hamster Ovary (CHO) cells.

[0319] PCR

[0320] A 5′ primer incorporating a consensus Kozak sequence was used inconjunction with a 3′ primer situated immediately upstream of thetransmembrane region to amplify the truncated cDNA. This product wasthen spliced into a pGEM-TEasy vector containing an Enterokinasecleavage site N-terminal to a Myc-His tag. The truncated c5T4_EKMycHiswas then cloned into pIRES neo.

[0321] This construct (pIRES_c5T4 Tm-)was transfected into CHO cells andstable lines made using G418 selection. Clones expressing thec5T4_EKMycHis were assessed by immunostaining with the Y1 chickenantiserum and an anti-His antibody.

[0322] Western blotting of cell supernatants with an anti-Myc antibodydemonstrates the presence of 5T4 in the cell supernatants of apIRES_c5T4 TM-stable transfectant (FIG. 5).

[0323] A suitable clone was then expanded and the supernatant harvested.The c5T4_EKMycHis protein was then isolated on a nickel column andpurity assessed by PAGE followed by coomassie staining and western blotanalysis.

[0324] Purification of c5T4TM-EKMycHis

[0325] c5T4TM-EKMycHis was double-purified from 1I CHO-c5T4TM-EKMycHissupernatant cells using a 5 ml HiTrap Chelating column and associatedHisTrap kit (Amersham Pharmacia Biotech) and the imidazole concentrationfrom elution was reduced by dialysis against 1× PBS (FIG. 6).

Example 4 Cloning Feline 5T4

[0326] The feline 5T4 gene was isolated from a feline genomic lambdalibrary, and following restriction analysis and Southern blotting, a 3kb DNA fragment was cloned into pBluescript (Stratagene) andsequenced—found to contain f5T4 gene. This was then amplified by PCR toclone both full-length and TM-f5T4 into expression vectors (pIRESneo)for transfection into CHO cells. The feline gene has also been clonedinto the appropriate MVA transfer plasmids in order to make recombinantMVA_f5T4.

[0327] Analysis of cloned cell line CHO-f5T4tm-reveals immunostainingidentical to that of CHO-c5T4tm-.

Example 5 Production of Recombinant MVA_c5T4

[0328] The canine 5T4 gene was cloned into the MVA vector and expressionof the canine 5T4 protein in cells infected with the modified virus wasdemonstrated. Expression of the canine protein in canine thymic cellswas also demonstrated and a stock of MVA_c5T4 vaccine prepared.

[0329] To demonstrate the ability of both feline and canine cells tosupport expression of 5T4 from recombinant MVA, western blot analysis ofthe following cells; C2fTH (Canine thymus), D17 (Poodle osteosarcoma),AK-D (Feline Foetal lung), CRFK (Feline Kidney), infected withrecombinant MVA_h5T4 was carried out. The cells were infected with MVAor MVA_(h)5T4 at an MOI of 5 and then harvested 24 hours later. Lysateswere subjected to PAGE and electroblotting to Hybond ECL and 5T4 wasthen detected with mAb H8 followed by RαM-HRP and ECL (FIG. 8).

Example 6 Cloning c5T4 into the MVA Transfer Vectors

[0330] Methods for propagation of MVA, preparation of CEF cells andhomologous recombination are described in WO 00/29428.

[0331] The full length c5T4 cDNA, amplified by PCR, was cloned into anMVA transfer plasmid with the LacZ marker gene (FIG. 9) which was thenused to make recombinant MVA_c5T4_LacZ by cotransfection with MVA intoCEFS. The recombinant virus has been purified to homogeneity through 4rounds of plaque picking, with no background of wild type virusdetected. Immunostaining of infected CEF cells with the Y1 chickenantiserum confirmed the presence of 5T4 in the virus and PCRamplification of a ˜350 bp fragment of 5T4 sequence from viral DNA hasconfirmed the 5T4 gene as canine.

Example 7 Induction of c5T4 expression in CEFs and canine thymic cells

[0332] Canine 5T4 was expressed in CEFs infected with recombinantMVA-c5T4. CEFs infected with MVA-c5T4lacZ (MOI=0.02) were immunostainedwith Y1 and Y3-P3 (FIG. 10) showing c5T4 expression.

[0333] Lysates from CEFs infected with MVA-c5T4lacZ (MOI=1) wereanalysed by Western Blot using Y1 (FIG. 11).

[0334] Having shown that the recombinant MVA_c5T4 expresses 5T4 proteinin CEFs it was also demonstrated that the protein could be expressed incanine cells. FIG. 12 shows a Western Blot of canine thymic (CF2TH)cells infected by MVA_c5T4lacZ and MVA_h5T4, detected using Y1.

Example 8 Immunohistochemistry of Canine and Feline Tissues

[0335] Normal canine tissues have been stained for 5T4 using the Y1antiserum, as shown below: Tissue No. Positive Brain 0/3 Cerebellum 0/3Heart 0/3 Lung 1/3 Liver 2/3 Kidney 3/3 Pituitary 3/3

[0336] The positive samples may be due to 5T4 expression but could bedue to some cross reactivity of Y1. To address the later possibility,the purified Y3-P3 has been used to repeat these studies of normaltissues. To date only some specific cells of the pituitary have stainedpositive with Y3-P3 whilst the cancer tissue samples that have beenstained with this antisera have remained positive.

[0337] A summary of the Y1 staining of canine and feline cancer samplesare given in tables 1 & 2. Out of all the different feline tumour typesstudied 23% (6/26) were positive. This rises to 38% (3/8) for mammarytumours only. The percentage of 5T4 positive tumours is higher in thecanine samples; 45% (30/66) for all types. When mammary tumours, whichmake up 42% of all the tumour types presented to Oncodesign, areanalysed separately, 75% (21/28) are 5T4 positive.

[0338]FIG. 13 shows two examples of 5T4 positive canine turmoursamples:mammary carcinoma (A) and anal apocrine carcinoma (B) TABLE 1FELINE SAMPLES Sex N^(o). +ve N^(o). −ve % +ve Mammary Tumors:-Adenocarcinoma M F 2 4 33 Lymphoma M F 1 0 Recurrence of canalicular(?)M F 1 100 TOTAL Mammary M F 3 5 38 Intra muscular fibromatosis M F 1 100Malignant Fibroblast M F 1 0 Bladder carcinoma M F 1 100 Sarcoma M F 1 0Fibrosarcoma M 2 0 F 6 0 Fibrohistocytoma M 1 0 F 4 ALL CANCERS (n = 26)M 3 0 F 6 17 26 Both 6 20 23

[0339] TABLE 2 CANINE SAMPLES Sex N^(o). +ve N^(o). −ve % +ve MammaryTumors:- (Adeno)carcinoma M F 15 3 83 Epithelial M F 5 1 83 MesenchymalM F 1 0 Intracanalicular(?) M F 1 100 Sarcoma M F 1 0 CutaneousMetastasis M F 1 0 TOTAL M F 21 7 75 Anal apocrine gland (+met.) M 1 0 F1 100 Intestinal Adenocarcinoma M F 1 100 Pre-cancerous mastopathy M F 21 67 Epidermoid Carcinoma M 1 100 F 1 100 Dermo-epidermic lesion M F 1100 Mast cell cancers M 3 0 F 2 0 Bladder carcinoma M F 1 1 50Osteosarcoma M 1 100 F Hepatocarcinoma M 1 0 F Seminoma M 2 0 F ThyroidCarcinoma M F 1 0 Lymphoma M 2 0 F 1 0 Myxosarcoma M 1 0 F FibrosarcomaM 1 0 F Fibrohistocytoma M F 1 0 Rhabdomyosarcoma M F 1 0 Malignantmelanoma M 5 0 F Malignant scwannoma M F 1 0 Hemangiopericytoma M 1 0 F2 0 Sertolinoma M 1 0 F ALL CANCERS (n = 66) M 2 18 10 F 28 18 61 Both30 36 45

[0340] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inchemistry, biology or related fields are intended to be within the scopeof the following claims.

1 12 1 420 PRT Canis sp. 1 Met Pro Gly Gly Cys Ser Arg Gly Pro Ala AlaGly Asp Gly Arg Leu 1 5 10 15 Arg Leu Ala Arg Leu Ala Leu Val Leu LeuGly Trp Val Ser Ser Ser 20 25 30 Ser Leu Thr Ser Trp Ala Pro Ser Ala AlaAla Ser Thr Ser Pro Pro 35 40 45 Ala Ser Ala Ala Ser Ala Pro Pro Pro LeuPro Gly Gln Cys Pro Gln 50 55 60 Pro Cys Glu Cys Ser Glu Ala Ala Arg ThrVal Lys Cys Val Asn Arg 65 70 75 80 Asn Leu Thr Glu Val Pro Ala Asp LeuPro Pro Tyr Val Arg Asn Leu 85 90 95 Phe Leu Thr Gly Asn Gln Leu Ala ValLeu Pro Pro Gly Ala Phe Ala 100 105 110 Arg Arg Pro Pro Leu Ala Glu LeuAla Ala Leu Asn Leu Ser Gly Ser 115 120 125 Ser Leu Arg Glu Val Cys AlaGly Ala Phe Glu His Leu Pro Ser Leu 130 135 140 Arg Gln Leu Asp Leu SerHis Asn Pro Leu Gly Asn Leu Ser Ala Phe 145 150 155 160 Ala Phe Ala GlySer Asp Ala Ser Arg Ser Gly Pro Ser Pro Leu Val 165 170 175 Glu Leu MetLeu Asn His Ile Val Pro Pro Asp Asp Arg Arg Gln Asn 180 185 190 Arg SerPhe Glu Gly Met Val Ala Ala Ala Leu Arg Ala Gly Arg Ala 195 200 205 LeuArg Gly Leu Gln Cys Leu Glu Leu Ala Gly Asn Arg Phe Leu Tyr 210 215 220Leu Pro Arg Asp Val Leu Ala Gln Leu Pro Gly Leu Arg His Leu Asp 225 230235 240 Leu Arg Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn245 250 255 Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu LysVal 260 265 270 Leu His Asn Ala Thr Leu Ala Glu Leu Gln Ser Leu Pro HisVal Arg 275 280 285 Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys HisMet Ala Asp 290 295 300 Met Val Ala Trp Leu Lys Glu Thr Glu Val Val ProGly Lys Ala Gly 305 310 315 320 Leu Thr Cys Ala Phe Pro Glu Lys Met ArgAsn Arg Ala Leu Leu Glu 325 330 335 Leu Asn Ser Ser His Leu Asp Cys AspPro Ile Leu Pro Pro Ser Leu 340 345 350 Gln Thr Ser Tyr Val Phe Leu GlyIle Val Leu Ala Leu Ile Gly Ala 355 360 365 Ile Phe Leu Leu Val Leu TyrLeu Asn Arg Lys Gly Ile Lys Lys Trp 370 375 380 Met His Asn Ile Arg AspAla Cys Arg Asp His Met Glu Gly Tyr His 385 390 395 400 Tyr Arg Tyr GluIle Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser Ser 405 410 415 Asn Ser AspVal 420 2 1263 DNA Canis sp. 2 atgcctgggg ggtgctcccg gggccccgccgccggggacg ggcggttgcg gctggcgcgg 60 ctggcgctgg tgctcctggg ctgggtctcctcgtcctcgc tcacctcctg ggcgccctcc 120 gccgccgcct ccacgtcgcc gccggcctccgcggcgtccg ccccgccccc gctgccgggc 180 cagtgccccc agccttgcga gtgctcggaggcggcgcgca cggtcaagtg cgttaaccgc 240 aacctgaccg aggtgcccgc ggacctgcccccctacgtgc gcaacctctt cctcacgggc 300 aaccagctgg cggtgctgcc ccccggcgccttcgcccgcc ggccgccgct ggccgagctg 360 gccgcgctca acctgagcgg cagcagcctgcgggaggtgt gcgccggcgc cttcgagcac 420 ctgcccagcc tgcgccagct cgacctcagccacaacccgc tgggcaacct cagcgccttc 480 gccttcgcgg gcagcgacgc cagccgctcgggccccagcc ccctggtgga gctgatgctg 540 aaccacatcg tgccccccga cgaccggcggcagaaccgga gcttcgaggg catggtggcg 600 gctgccctcc gagcgggccg cgcgcttcgcgggctgcagt gcctggagct ggccggcaac 660 cgcttcctct acttgcctcg cgacgtcctggcccagctac ccggcctccg gcacctggac 720 ctgcgcaaca actccctggt gagcctcacctacgtgtcct tccgcaacct gacgcacttg 780 gagagcctcc acctggagga caacgccctcaaggtccttc acaacgccac cctggcggag 840 ctgcagagcc tgccccacgt ccgggtcttcctggacaaca acccctgggt ctgcgattgt 900 cacatggcag acatggtggc ctggctcaaggagacagagg tggtgccggg caaagccggg 960 ctcacctgtg cattcccgga gaaaatgaggaatcgggccc tcttggaact caacagctcc 1020 cacctggact gtgaccctat cctccctccatccctgcaga cttcttatgt cttcctaggt 1080 attgtcttag ccctgatagg cgccatcttcctactggttt tgtatttgaa ccgcaagggg 1140 ataaagaagt ggatgcataa catcagagatgcctgcaggg atcacatgga agggtatcac 1200 tacagatacg aaatcaatgc agaccccaggttaacaaacc tcagttccaa ttcggatgtc 1260 tga 1263 3 419 PRT Felis sp. 3 MetPro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly Asp Gly Arg Leu 1 5 10 15Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ser Ser 20 25 30Ser Leu Thr Ser Ser Ala Pro Ser Thr Ser Ser Thr Ser Phe Leu Ala 35 40 45Ser Ala Val Ser Ala Gln Pro Pro Leu Pro Gly Gln Cys Pro Gln Leu 50 55 60Cys Glu Cys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg Asn 65 70 7580 Leu Thr Glu Val Pro Ala Asp Leu Pro Pro Tyr Val Arg Asn Leu Phe 85 9095 Leu Thr Gly Asn Gln Leu Ala Val Leu Pro Ala Gly Ala Phe Ala Arg 100105 110 Arg Pro Pro Leu Ala Glu Leu Ala Ala Leu Asn Leu Ser Gly Ser Arg115 120 125 Leu Gln Glu Val Arg Ala Gly Ala Phe Glu Gln Leu Pro Ser LeuArg 130 135 140 Gln Leu Asp Leu Ser His Asn Pro Leu Ala His Leu Ser ProPhe Thr 145 150 155 160 Phe Ser Gly Ser Asn Ala Ser Phe Ser Ala Pro SerPro Leu Val Glu 165 170 175 Leu Met Leu Asn His Ile Val Pro Pro Glu AspHis Arg His Asn Arg 180 185 190 Ser Phe Glu Gly Met Val Ala Ala Ser LeuArg Ala Gly His Ala Leu 195 200 205 Arg Gly Leu Gln Arg Leu Glu Leu AlaSer Asn His Phe Leu Phe Leu 210 215 220 Pro Arg Asp Val Leu Ala His LeuPro Gly Leu Arg His Leu Asp Leu 225 230 235 240 Arg Asn Asn Ser Leu ValSer Leu Thr Tyr Val Ser Phe Arg Asn Leu 245 250 255 Thr His Leu Gln SerLeu His Leu Glu Asp Asn Ala Leu Lys Val Leu 260 265 270 His Asn Gly ThrMet Ala Glu Leu Gln Ser Leu Pro His Val Arg Val 275 280 285 Phe Leu AspAsn Asn Pro Trp Val Cys Asp Cys His Met Val Asp Met 290 295 300 Val AlaTrp Leu Lys Glu Thr Glu Val Val Gln Gly Lys Ala Arg Leu 305 310 315 320Ala Cys Ala Phe Pro Glu Lys Met Arg Asn Arg Ala Leu Leu Glu Leu 325 330335 Asn Ser Ser His Leu Glu Cys Asp Pro Ile Leu Pro Pro Ser Leu Gln 340345 350 Thr Ser Tyr Val Phe Leu Gly Ile Val Leu Ala Leu Ile Gly Ala Ile355 360 365 Phe Leu Leu Val Leu Tyr Leu Asn Arg Lys Gly Ile Lys Lys TrpMet 370 375 380 His Asn Ile Arg Asp Ala Cys Arg Asp His Met Glu Gly TyrHis Tyr 385 390 395 400 Arg Tyr Glu Ile Asn Ala Asp Pro Arg Leu Thr AsnLeu Ser Ser Asn 405 410 415 Ser Asp Val 4 1260 DNA Felis sp. 4atgcctgggg ggtgctcccg gggccccgcc gccggagacg ggcggctgcg gctggcgcgg 60ctggcgctgg tcctcctggg ctgggtctct tcgtcttctc tcacttcctc ggcgccctcc 120acctcctcca cgtcgttcct ggcctccgcg gtgtccgccc agcccccgct gccgggccaa 180tgcccccagc tttgcgagtg ctccgaggcg gcgcgcactg tcaagtgcgt taaccgcaac 240ctgaccgagg tgcccgcgga cctgcccccc tacgtgcgca acctcttcct caccggcaat 300cagctggccg tgctccccgc cggcgccttc gcccgccggc cgccgctggc ggagctggcc 360gcgctcaacc tcagcggcag ccgcctgcag gaggtgcgcg ccggcgcctt cgagcaactg 420cccagcctgc ggcagctcga cctcagccac aacccgctgg cccacctcag ccccttcacc 480ttctcgggca gcaacgccag cttctcggcc cccagccccc tggtggaact gatgctgaac 540cacatcgtgc cccctgagga ccaccggcac aaccggagct tcgagggtat ggtggcggcg 600tccctacgcg ccggccatgc gcttcgcggg ctccagcgcc tygaactggc cagcaaccac 660ttcctcttct tgcctcggga cgtactggcc cacctaccgg gcctcaggca cctggacctg 720cgcaacaact cgctggtgag cctaacttac gtgtccttcc gcaacctgac acacctacaa 780agcctccacc tggaggacaa cgccctcaag gtccttcaca acggcaccat ggcggagttg 840cagagcctgc cccacgtcag ggtcttcctg gacaacaatc cctgggtctg cgactgtcac 900atggtggaca tggtggcctg gctcaaggag acagaggtag tgcagggcaa agccaggctc 960gcctgtgcat tcccggaaaa aatgaggaat cgggcccttt tggaactcaa cagctcccac 1020ctggagtgtg accctatcct ccctccatcc ctgcagactt cttatgtctt tctaggtatt 1080gttttagccc tgataggtgc cattttctta ctggttttgt acttgaaccg caaggggata 1140aaaaagtgga tgcataacat cagagatgcc tgcagggatc acatggaagg gtatcactac 1200agatatgaaa tcaacgcgga ccccaggtta acaaacctca gttctaattc ggatgtctga 1260 534 DNA Canis sp. 5 cccagctccg ggagcgccgc gccgcgccgc gatg 34 6 21 DNAHomo sapiens 6 agctccgggg aaacgcgagc c 21 7 30 DNA Homo sapiens 7cccagctccg gggaaacgcg agccgcgatg 30 8 20 PRT Artificial sequence 5T4peptide for antibody production, described in Example 1 8 Cys Arg TyrGlu Ile Asn Ala Asp Pro Arg Leu Thr Asn Leu Ser Ser 1 5 10 15 Asn SerAsp Val 20 9 19 PRT Artificial sequence 5T4 peptide for antibodyproduction, described in Example 1 9 Cys Leu Asn His Ile Val Pro Pro GluAsp Glu Arg Gln Asn Arg Ser 1 5 10 15 Phe Glu Gly 10 23 PRT Artificialsequence 5T4 peptide for antibody production, described in Example 1 10Asn Leu Ser Gly Ser Arg Leu Asp Glu Val Arg Ala Gly Ala Phe Glu 1 5 1015 His Leu Pro Ser Leu Arg Cys 20 11 426 PRT Mus sp. 11 Met Pro Gly AlaGly Ser Arg Gly Pro Ser Ala Gly Asp Gly Arg Leu 1 5 10 15 Arg Leu AlaArg Leu Ala Leu Val Leu Leu Gly Trp Val Ser Ala Ser 20 25 30 Ala Pro SerSer Ser Val Pro Ser Ser Ser Thr Ser Pro Ala Asp Phe 35 40 45 Leu Ala SerGly Ser Ala Gln Pro Pro Pro Ala Glu Arg Cys Pro Ala 50 55 60 Ala Cys GluCys Ser Glu Ala Ala Arg Thr Val Lys Cys Val Asn Arg 65 70 75 80 Asn LeuLeu Glu Val Pro Ala Asp Leu Pro Pro Tyr Val Arg Asn Leu 85 90 95 Phe LeuThr Gly Asn Gln Met Thr Val Leu Pro Ala Gly Ala Phe Ala 100 105 110 ArgGln Pro Pro Leu Ala Asp Leu Glu Ala Leu Asn Leu Ser Gly Asn 115 120 125His Leu Lys Glu Val Cys Ala Gly Ala Phe Glu His Leu Pro Gly Leu 130 135140 Arg Arg Leu Asp Leu Ser His Asn Pro Leu Thr Asn Leu Ser Ala Phe 145150 155 160 Val Phe Ala Gly Ser Asn Ala Ser Val Ser Ala Pro Ser Pro LeuGlu 165 170 175 Glu Leu Ile Leu Asn His Ile Val Pro Pro Glu Asp Gln ArgGln Asn 180 185 190 Gly Ser Phe Glu Gly Met Val Ala Phe Glu Gly Met ValAla Ala Ala 195 200 205 Leu Arg Ser Gly Leu Ala Leu Arg Gly Leu Thr ArgLeu Glu Leu Ala 210 215 220 Ser Asn His Phe Leu Phe Leu Pro Arg Asp LeuLeu Ala Gln Leu Pro 225 230 235 240 Ser Leu Arg Tyr Leu Asp Leu Arg AsnAsn Ser Leu Val Ser Leu Thr 245 250 255 Tyr Ala Ser Phe Arg Asn Leu ThrHis Leu Glu Ser Leu His Leu Glu 260 265 270 Asp Asn Ala Leu Lys Val LeuHis Asn Ser Thr Leu Ala Glu Trp Gln 275 280 285 Gly Leu Ala His Val LysVal Phe Leu Asp Asn Asn Pro Trp Val Cys 290 295 300 Asp Cys Tyr Met AlaAsp Met Val Ala Trp Leu Lys Glu Thr Glu Val 305 310 315 320 Val Pro AspLys Ala Arg Leu Thr Cys Ala Phe Pro Glu Lys Met Arg 325 330 335 Asn ArgGly Leu Leu Asp Leu Asn Ser Ser Asp Leu Asp Cys Asp Ala 340 345 350 ValLeu Pro Gln Ser Leu Gln Thr Ser Tyr Val Phe Leu Gly Ile Val 355 360 365Leu Ala Leu Ile Gly Ala Ile Phe Leu Leu Val Leu Tyr Leu Asn Arg 370 375380 Lys Gly Ile Lys Lys Trp Met His Asn Ile Arg Asp Ala Cys Arg Asp 385390 395 400 His Met Glu Gly Tyr His Tyr Arg Tyr Glu Ile Asn Ala Asp ProArg 405 410 415 Leu Thr Asn Leu Ser Ser Asn Ser Asp Val 420 425 12 420PRT Homo sapiens 12 Met Pro Gly Gly Cys Ser Arg Gly Pro Ala Ala Gly AspGly Arg Leu 1 5 10 15 Arg Leu Ala Arg Leu Ala Leu Val Leu Leu Gly TrpVal Ser Ser Ser 20 25 30 Ser Pro Thr Ser Ser Ala Ser Ser Phe Ser Ser SerAla Pro Phe Leu 35 40 45 Ala Ser Ala Val Ser Ala Gln Pro Pro Leu Pro AspGln Cys Pro Ala 50 55 60 Leu Cys Glu Cys Ser Glu Ala Ala Arg Thr Val LysCys Val Asn Arg 65 70 75 80 Asn Leu Thr Glu Val Pro Thr Asp Leu Pro AlaTyr Val Arg Asn Leu 85 90 95 Phe Leu Thr Gly Asn Gln Leu Ala Val Leu ProAla Gly Ala Phe Ala 100 105 110 Arg Arg Pro Pro Leu Ala Glu Leu Ala AlaLeu Asn Leu Ser Gly Ser 115 120 125 Arg Leu Asp Glu Val Arg Ala Gly AlaPhe Glu His Leu Pro Ser Leu 130 135 140 Arg Gln Leu Asp Leu Ser His AsnPro Leu Ala Asp Leu Ser Pro Phe 145 150 155 160 Ala Phe Ser Gly Ser AsnAla Ser Val Ser Ala Pro Ser Pro Leu Val 165 170 175 Glu Leu Ile Leu AsnHis Ile Val Pro Pro Glu Asp Glu Arg Gln Asn 180 185 190 Arg Ser Phe GluGly Met Val Val Ala Ala Leu Leu Ala Gly Arg Ala 195 200 205 Leu Gln GlyLeu Arg Arg Leu Glu Leu Ala Ser Asn His Phe Leu Tyr 210 215 220 Leu ProArg Asp Val Leu Ala Gln Leu Pro Ser Leu Arg His Leu Asp 225 230 235 240Leu Ser Asn Asn Ser Leu Val Ser Leu Thr Tyr Val Ser Phe Arg Asn 245 250255 Leu Thr His Leu Glu Ser Leu His Leu Glu Asp Asn Ala Leu Lys Val 260265 270 Leu His Asn Gly Thr Leu Ala Glu Leu Gln Gly Leu Pro His Ile Arg275 280 285 Val Phe Leu Asp Asn Asn Pro Trp Val Cys Asp Cys His Met AlaAsp 290 295 300 Met Val Thr Trp Leu Lys Glu Thr Glu Val Val Gln Gly LysAsp Arg 305 310 315 320 Leu Thr Cys Ala Tyr Pro Glu Lys Met Arg Asn ArgVal Leu Leu Glu 325 330 335 Leu Asn Ser Ala Asp Leu Asp Cys Asp Pro IleLeu Pro Pro Ser Leu 340 345 350 Gln Thr Ser Tyr Val Phe Leu Gly Ile ValLeu Ala Leu Ile Gly Ala 355 360 365 Ile Phe Leu Leu Val Leu Tyr Leu AsnArg Lys Gly Ile Lys Lys Trp 370 375 380 Met His Asn Ile Arg Asp Ala CysArg Asp His Met Glu Gly Tyr His 385 390 395 400 Tyr Arg Tyr Glu Ile AsnAla Asp Pro Arg Leu Thr Asn Leu Ser Ser 405 410 415 Asn Ser Asp Val 420

1. A canine 5T4 polypeptide having the amino acid sequence shown in SEQID No 1 or a variant, homologue, fragment or derivative thereof.
 2. Anucleotide sequence capable of encoding a canine 5T4 polypeptideaccording to claim
 1. 3. A nucleotide sequence according to claim 2,having the sequence shown as SEQ ID NO 2 or a variant, homologue,fragment or derivative thereof.
 4. A feline 5T4 polypeptide having theamino acid sequence shown in SEQ ID No 3 or a variant, homologue,fragment or derivative thereof.
 5. A nucleotide sequence capable ofencoding a feline 5T4 polypeptide according to claim
 4. 6. A nucleotidesequence according to claim 5, having the sequence shown as SEQ ID NO 4or a variant, homologue, fragment or derivative thereof.
 7. A vectorsystem which comprises a polynucleotide encoding a canine or feline 5T4antigen.
 8. A vector system according to claim 7, wherein thepolynucleotide encodes a 5T4 polypeptide or a variant, homologue,fragment or derivative thereof according to claim 1 or
 3. 9. A vectorsystem according to claim 7 or 8, which is an MVA vector system.
 10. Anagent specific for a canine or feline 5T4 polypeptide according to claim1 or 3, or a variant, homologue, fragment or derivative thereof.
 11. Anagent according to claim 10 which is an antibody.
 12. An agent accordingto claim 11 which is an ScFv antibody.
 13. A kit comprising a vectorsystem according to any of claims 7 or 8 and an agent according to anyof claims 10 to 12 or a precursor thereof for simultaneous, separate orsequential administration to a subject
 14. A vaccine, a primingcomposition or a boosting composition which comprises a polypeptide (ora variant, homologue, fragment or derivative thereof) according to claim1 or 4, a polynucleotide (or a variant, homologue, fragment orderivative thereof) according to claim 2, 3, 5 or 6, a vector systemaccording to any of claims 7 or 8, or an agent according to any ofclaims 10 to
 12. 15. A vaccination kit which comprises: a firstcomposition comprising a polynucleotide encoding a canine or feline 5T4antigen, and a second composition comprising a vector system accordingto any of claims 7 or 8 for simultaneous, separate or sequentialadministration to a subject.
 16. A kit according to claim 15, whereinthe second composition comprises a polynucleotide according to claim 2,3, 5 or
 6. 17. The use of a vaccine, a priming composition or a boostingcomposition according to claim 14, or a kit according to claim 15 or 16in the manufacture of a medicament for the treatment and/or preventionof a disease in a subject.
 18. A method for treating and/or preventing adisease in a subject which comprises the step of administering avaccine, a priming composition or a boosting composition according toclaim 14, or a kit according to claim 15 or 16 to the subject.
 19. Theuse according to claim 17 or the method according to claim 18, whereinthe disease is cancer.